Pdgf receptor beta binding polypeptides

ABSTRACT

The present invention provides binding polypeptides (e.g., antibodies or fragments thereof) that specifically bind to a target antigen (e.g., a human antigen, e.g., human PDGFRβ) with high affinity. The invention also provides, libraries of binding polypeptides, pharmaceutical compositions, as well as nucleic acids encoding binding polypeptides, recombinant expression vectors and host cells for making such binding polypeptides. Methods of using binding polypeptide of the invention to diagnose and treat disease are also encompassed by the invention.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/792,728, filed Feb. 17, 2020, which is a continuation of U.S. patentapplication Ser. No. 15/207,188, filed Jul. 11, 2016, now U.S. Pat. No.10,604,578, which is a continuation of U.S. patent application Ser. No.13/705,978, filed Dec. 5, 2012, now U.S. Pat. No. 9,416,179, whichclaims priority to U.S. Provisional Patent Application Serial Nos.61/566,778, filed Dec. 5, 2011; and 61/610,905, filed Mar. 14, 2012, allof which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 24, 2021, isnamed 721569_XBI-005CON3_ST25.txt and is 242,599 bytes in size.

INTRODUCTION

Platelet derived growth factor (PDGF) is a potent mitogen andchemoattractant in cells of mesenchymal origin and is involved in thepathologies of many diseases. PDGF exists as a disulfide-linked dimerconsisting of two homologous chains, A or B, that can combine to formthree distinct PDGF isoforms, AA, BB or AB. All isoforms of PDGF mediatetheir mitogenic effect by binding to a cell surface PDGF receptor(PDGFR).

PDGF receptors belong to the tyrosine kinase family and consist of tworeceptor isoforms, alpha and beta. The alpha and beta isoforms can bedistinguished by their distinct ligand binding specificities. PDGF betareceptor can bind to only B-chain (isoforms BB and AB), while PDGF alphareceptor can bind to all isoforms of PDGF.

Binding of PDGF to a cell surface PDGFR causes receptor dimerization andtrans-autophosphorylation which, in turn, results in intracellularsignalling events that cause, inter alia, cell proliferation and cellmigration. Accordingly, PDGFR antagonists that block PDGF binding and/orreceptor dimerization can be used to treat or prevent diseasesassociated with PDGFR activation.

There is a therefore a need in the art for novel PDGFR antagonists thatcan be used to treat diseases associated with PDGFR activation.

SUMMARY OF THE INVENTION

The present invention provides binding polypeptides (e.g., antibodies orfragments thereof) that specifically bind to a target antigen (e.g., ahuman antigen, e.g., human PDGF) with high affinity. In a preferredembodiment, the invention provides binding polypeptides that bind toPDGFRβ (e.g., human PDGFRβ) with high affinity and antagonize PDGFRβactivation. Such binding polypeptides are particularly useful fortreating PDGFRβ-associated diseases or disorders (e.g., age-relatedmacular degeneration (AMD)). The invention also provides, libraries ofbinding polypeptides, pharmaceutical compositions, as well as nucleicacids encoding binding polypeptides, recombinant expression vectors andhost cells for making such binding polypeptides. Methods of usingbinding polypeptides of the invention to detect PDGFRβ and to modulatePDGFRβ activity are also encompassed by the invention.

Accordingly, in one aspect the invention provides an isolated bindingpolypeptide comprising a VH domain, wherein, as an isolated domain, theVH domain binds to an antigen with a Kd of less than 100 pM.

In another aspect, the invention provides an isolated bindingpolypeptide that specifically binds to PDGFRβ, comprising the CDR3sequence set forth in SEQ ID NO: 1.

In certain embodiments, the binding polypeptide comprises a VH domaincomprising the HCDR3 amino acid sequence set forth in SEQ ID NO: 1. TheVH domain may further comprise a HCDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2-32, and/or a HCDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 33-62.

In certain embodiments, the polypeptide comprises a VH domain comprisingan amino acid sequence sharing at least 80% (e.g., 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99%) amino acid identity with a VH domain amino acid sequenceselected from the group consisting of SEQ ID NOs: 318-368.

In certain embodiments, the polypeptide comprises a VH domain comprisingan am amino acid sequence selected from the group consisting of SEQ IDNOs: 318-368

In certain embodiments, the binding polypeptide comprises a VL domain.The VH domain may further comprise a LCDR3 comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 63-147, aLCDR2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 148-232, and/or a LCDR1 comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 233-317.

In certain embodiments, the polypeptide comprises a VL domain comprisingan amino acid sequence sharing at least 80% (e.g., 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99%) amino acid identity with a VL domain amino acid sequenceselected from the group consisting of SEQ ID NOs: 369-453.

In certain embodiments, the polypeptide comprises a VL domain comprisingan amino acid sequence selected from the group consisting of SEQ ID NOs:369-453.

In further aspect, the invention provides a binding polypeptide thatbinds to the same epitope on PDGFRβ as a binding polypeptide comprisingthe VH domain amino acid sequence set forth in SEQ ID No: 318. In apreferred embodiment, the binding polypeptide comprises a VH domainamino acid sequence sharing at least 80% amino acid identity (e.g., 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99%) with a VH domain amino acid sequenceselected from the group consisting of SEQ ID NOs: 318-368.

In a further aspect, the invention provides a binding polypeptide thatcompetes for binding to PDGFRβ with a binding polypeptide comprising theVH domain amino acid sequence set forth in SEQ ID No: 318. In apreferred embodiment, the binding polypeptide comprises a VH domainamino acid sequence sharing at least 80% amino acid identity with a VHdomain amino acid sequence selected from the group consisting of SEQ IDNOs: 318-368.

In certain embodiments, the binding polypeptides of the inventioninhibit the activity of PDGFRβ. In one embodiment, the activity ofPDGFRβ is inhibited by antagonizing PDGF binding to PDGFRβ. In anotherembodiment, the activity of PDGFRβ is inhibited by antagonizing PDGFRβdimerization.

In certain embodiments, the binding polypeptides of the invention bindto PDGFRβ with a Kd of less than 100 pM and/or with an off-rate of lessthan 10⁻³ s⁻¹.

In certain embodiments, the binding polypeptides of the invention bindspecifically to mouse and human PDGFRβ.

In certain embodiments, the binding polypeptides of the inventionantagonize PDGF binding to the PDGFRβ with an IC50 of less than 5 nM,inhibit ligand induced tyrosine phosphorylation of PDGFRβ with an IC50of less than 4 nM, inhibit retinal pericyte migration with an IC50 ofless than 6 nM, and/or have a melting temperature (Tm) of at least 68°C.

In a further aspect, the invention provides an isolated nucleic acidencoding a binding polypeptide of the invention.

In a further aspect, the invention provides a recombinant expressionvector comprising an isolated nucleic acid encoding a bindingpolypeptide of the invention.

In a further aspect, the invention provides a host cell expressing abinding polypeptide of the invention.

In a further aspect, the invention provides a method of producing abinding polypeptide that binds specifically to human PDGFRβ, comprisingculturing a host cell capable of expressing a binding polypeptide of theinvention under conditions such that the binding polypeptide is producedby the host cell.

In a further aspect, the invention provides a pharmaceutical compositioncomprising a binding polypeptide of the invention and one or morepharmaceutically acceptable carrier.

In a further aspect, the invention provides a method for treating adisease or disorder PDGFRβ-associated disease or disorder (e.g.,age-related macular degeneration (AMD) or cancer), the method comprisingadministering to a subject in need of thereof a pharmaceuticalcomposition of the invention.

In a further aspect, the invention provides a diverse library ofunpaired VH domains wherein each member of the library binds to humanPDGFRβ. In one preferred embodiment, each member of the librarycomprises the CDR3 amino acid sequence set forth in SEQ ID NO: 1 anddiversity lies in the FR1-FR3 regions. In one preferred embodiment, thelibrary is a nucleic acid display library (e.g., a DNA display library).

In a further aspect, the invention provides a diverse library of stableVH/VL pairs wherein each member of the library binds to human PDGFRβ. Inone preferred embodiment, each member of the library comprises a VHdomain comprising the CDR3 amino acid sequence set forth in SEQ IDNO: 1. In one preferred embodiment, the VL domains are human VL domains.In one preferred embodiment, the library is a nucleic acid displaylibrary (e.g., a DNA display library).

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic representation of the construction of exemplary VHdomain nucleic acid display libraries for use in the disclosed methods.

FIG. 2 depicts the results of in vitro binding assays measuring thebinding to human or mouse PDGFRβ of the XB1511VH domain, an unselectedXB1511 CDR3/framework shuffled DNA display library (RO), and an XB1511CDR3/framework shuffled DNA display library pool after four rounds ofselection (R4).

FIG. 3 is a schematic representation of the construction of exemplary VLnucleic acid display libraries for use in the disclosed methods.

FIG. 4 depicts the results of ELISA assays measuring the binding tohuman PDGFRβ of XB1511/VL scFv comprising VL domains isolated from thesecond round screening pool of a VH/VL pairing DNA display screen.

FIG. 5 depicts the results of ELISA assays measuring the binding tohuman PDGFRβ of XB1511/VL scFv comprising VL domains isolated from thethird round screening pool of a VH/VL pairing DNA display screen.

FIG. 6 depicts the results of ELISA assays measuring the binding tohuman PDGFRβ of XB2202/VL scFv comprising VL domains isolated from thesecond round screening pool of a VH/VL pairing DNA display screen.

FIG. 7 depicts the results of ELISA assays measuring the binding tohuman PDGFRβ of unpaired VL domains from the XB1511/VL scFv set forth inFIG. 9.

FIG. 8 depicts the results of solution binding affinity studiesmeasuring the binding to human PDGFRβ of ³⁵S Met labeled XB1511 VHdomain and XB1511-containing scFV obtained from VH/VL pairing DNAdisplay screens.

FIG. 9 depicts the results of solution binding affinity studiesmeasuring the binding to human PDGFRβ of ³⁵S Met labeled XB2202 VHdomain and XB2202-containing scFV obtained from VH/VL pairing DNAdisplay screens.

FIG. 10 depicts the results of surface Plasmon resonance binding studiesmeasuring the binding kinetics of XB1511 and the framework shuffledderivatives XB2202 and XB2708 to human PDGFRβ.

FIG. 11A depicts the results of surface plasmon resonance binding assaysmeasuring the binding kinetics of XB2202 to human PDGFRβ; FIG. 11Bdepicts the results of surface plasmon resonance binding assaysmeasuring the binding kinetics of XB2202 to mouse PDGFRβ.

FIG. 12A depicts the results of surface plasmon resonance binding assaysmeasuring the binding kinetics of XB2708 to human PDGFRβ; FIG. 12Bdepicts the results of surface plasmon resonance binding assaysmeasuring the binding kinetics of XB2708 to mouse PDGFRβ.

FIG. 13 depicts the results of surface plasmon resonance competitionbinding assays measuring the kinetics of binding of PDGF-BB to PDGFRβ atvarious concentrations of XB2202.

FIG. 14 depicts the results of in vitro cell migration assays measuringinhibition of pericyte migration by XB2708.

FIG. 15 depicts the results of label-free migration assays measuring theability of an XB1511-containing IgG1 to inhibit the migration of humanforeskin fibroblasts.

FIG. 16 depicts the results of ELISA assays measuring the binding tohuman PDGFRβ of XB2202 VH domain and XB2202/A4 scFv after incubation atvarious temperatures.

DETAILED DESCRIPTION

The present invention provides binding polypeptides (e.g., antibodies orfragments thereof) that specifically bind to a target antigen (e.g., ahuman antigen) with high affinity. In a preferred embodiment, thebinding polypeptides of the invention bind to PDGFRβ (e.g., humanPDGFRβ) with high affinity and inhibit the activity of PDGFRβ. Suchbinding polypeptides are particularly useful for treatingPDGFRβ-associated diseases or disorders (e.g., age-related maculardegeneration (AMD)). The invention also provides, libraries of bindingpolypeptides, pharmaceutical compositions, as well as nucleic acidsencoding binding polypeptides, recombinant expression vectors and hostcells for making such binding polypeptides. Methods of using bindingpolypeptides of the invention to detect PDGFRβ and to modulate PDGFRβactivity are also encompassed by the invention.

I. DEFINITIONS

In order that the present invention may be more readily understood,certain terms are first defined.

As used herein, the term “PDGFRβ” refers to platelet-derived growthfactor receptor beta. PDGFRβ nucleotide and polypeptide sequences arewell known in the art. An exemplary human PDGFRβ amino sequence is setforth in GenBank deposit GI:4505683 and an exemplary mouse PDGFRβ aminosequence is set forth in GenBank deposit GI: 226371752.

As used herein, the term “PDGF” refers to platelet-derived growthfactor. PDGF nucleotide and polypeptide sequences are well known in theart. An exemplary human PDGF amino sequence is set forth in GenBankdeposit GI:4505681 and an exemplary mouse PDGF amino sequence is setforth in GenBank deposit GI:400744.

As used herein, the term “binding polypeptide” refers to a polypeptidethat contains all or a part of the antigen binding site of an antibody(e.g., all or part of the heavy and/or light chain variable domain,e.g., at least HCDR3 of the heavy chain variable domain) such that thebinding polypeptide specifically recognizes a target antigen.Non-limiting examples of binding polypeptides include antibodies, orfragments thereof, and immunoglobulin-like domains (e.g., fibronectindomains) that have been altered to comprise all or a part of the antigenbinding site of an antibody.

As used herein, the term “antibody” refers to immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds, as well as multimersthereof (e.g., IgM). Each heavy chain comprises a heavy chain variableregion (abbreviated VH) and a heavy chain constant region. The heavychain constant region comprises three domains, CH1, CH2 and CH3. Eachlight chain comprises a light chain variable region (abbreviated VL) anda light chain constant region. The light chain constant region comprisesone domain (CL1). The VH and VL regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDRs), interspersed with regions that are more conserved, termedframework regions (FR).

As used herein, the term “antigen-binding portion” of an antibodyincludes any naturally occurring, enzymatically obtainable, synthetic,or genetically engineered polypeptide or glycoprotein that specificallybinds an antigen to form a complex. Antigen-binding fragments of anantibody may be derived, e.g., from full antibody molecules using anysuitable standard techniques such as proteolytic digestion orrecombinant genetic engineering techniques involving the manipulationand expression of DNA encoding antibody variable and optionally constantdomains. Non-limiting examples of antigen-binding portions include: (i)Fab fragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR)). Other engineered molecules,such as diabodies, triabodies, tetrabodies and minibodies, are alsoencompassed within the expression “antigen-binding portion.”

As used herein, the terms “VH domain” and “VL domain” refer to singleantibody variable heavy and light domains, respectively, comprising FR(Framework Regions) 1, 2, 3 and 4 and CDR (Complementary DeterminantRegions) 1, 2 and 3 (see Kabat et al. (1991) Sequences of Proteins ofImmunological Interest. (NIH Publication No. 91-3242, Bethesda).

As used herein, the term “FR1-FR3” refers to the region of a VHencompassing FR1, CDR2, FR2, CDR2 and FR3, but excluding the CDR3 andFR4 regions.

As used herein, the term “unpaired” refers to VH or VL that are notlinked (either covalently or non-covalently) to a complementary VL or VHdomain, respectively.

As used herein, the term “complementary VL or VH domain” refers to a VLor VH domain that associates with a VH or VL domain, respectively, toform a VH/VL pair.

As used herein, the term “VH/VL pair” refers to a non-covalent dimer ofa single VH and a single VL domain, wherein the VL and VH domain areassociated in a similar manner to that observed in a complete,tetrameric immunogobulin molecule, and the dimer can bind specificallyto at least one target antigen. A “stable VH/VL pair” is a VH/VL pairthat does not exhibit significant dissociation of the substituent VH andVL domains under physiological conditions.

As used herein, the term “CDR” or “complementarity determining region”means the noncontiguous antigen combining sites found within thevariable region of both heavy and light chain polypeptides. Theseparticular regions have been described by Kabat et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein ofimmunological interest. (1991), and by Chothia et al., J. Mol. Biol.196:901-917 (1987) and by MacCallum et al., J. Mol. Biol. 262:732-745(1996) where the definitions include overlapping or subsets of aminoacid residues when compared against each other. The amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth for comparison. Preferably, the term “CDR” is aCDR as defined by Kabat, based on sequence comparisons.

As used herein the term “framework (FR) amino acid residues” refers tothose amino acids in the framework region of an immunogobulin chain. Theterm “framework region” or “FR region” as used herein, includes theamino acid residues that are part of the variable region, but are notpart of the CDRs (e.g., using the Kabat definition of CDRs).

As used herein, the term “specifically binds to” refers to the abilityof a binding polypeptide to bind to an antigen with an Kd of at leastabout 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M,1×10⁻¹² M, or more, and/or bind to an antigen with an affinity that isat least two-fold greater than its affinity for a nonspecific antigen.It shall be understood, however, that the binding polypeptide arecapable of specifically binding to two or more antigens which arerelated in sequence. For example, the binding polypeptides of theinvention can specifically bind to both human and a non-human (e.g.,mouse or non-human primate) PDGFRβ.

As used herein, the term “antigen” refers to the binding site or epitoperecognized by a binding polypeptide.

As used herein, the term “nucleic acid display library” refers to anyart recognized in vitro cell-free phenotype-genotype linked display,including, without limitation those set forth in, for example, U.S. Pat.Nos. 7,195,880; 6,951,725; 7,078,197; 7,022,479; 6,518,018; 7,125,669;6,846,655; 6,281,344; 6,207,446; 6,214,553; 6,258,558; 6,261,804;6,429,300; 6,489,116; 6,436,665; 6,537,749; 6,602,685; 6,623,926;6,416,950; 6,660,473; 6,312,927; 5,922,545; and 6,348,315, and inWO2010/011944, which are all hereby incorporated by reference in theirentirety.

As used herein, the term “vector” is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. The terms, “plasmid” and “vector” may be usedinterchangeably. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

As used herein, the term “host cell” is intended to refer to a cell intowhich a recombinant expression vector has been introduced. It should beunderstood that this term is intended to refer not only to theparticular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

As used herein, the term “treat,” “treating,” and “treatment” refer totherapeutic or preventative measures described herein. The methods of“treatment” employ administration to a subject, an antibody or antigenbinding portion of the present invention, for example, a subject havinga PDGFRβ-associated disease or disorder (e.g. AMD) or predisposed tohaving such a disease or disorder, in order to prevent, cure, delay,reduce the severity of, or ameliorate one or more symptoms of thedisease or disorder or recurring disease or disorder, or in order toprolong the survival of a subject beyond that expected in the absence ofsuch treatment.

As used herein, the term “PDGFRβ-associated disease or disorder”includes disease states and/or symptoms associated with PDGFRβ activity.Exemplary PDGFRβ-associated diseases or disorders include, but are notlimited to, age-related macular degeneration (AMD) and cancer.

As used herein, the term “effective amount” refers to that amount of abinding polypeptide that is sufficient to effect treatment, prognosis ordiagnosis of a PDGFRβ-associated disease or disorder, as describedherein, when administered to a subject. A therapeutically effectiveamount will vary depending upon the subject and disease condition beingtreated, the weight and age of the subject, the severity of the diseasecondition, the manner of administration and the like, which can readilybe determined by one of ordinary skill in the art. The dosages foradministration can range from, for example, about 1 ng to about 10,000mg, about 1 ug to about 5,000 mg, about 1 mg to about 1,000 mg, about 10mg to about 100 mg, of a binding polypeptide according to the invention.Dosage regiments may be adjusted to provide the optimum therapeuticresponse. An effective amount is also one in which any toxic ordetrimental effects (i.e., side effects) of a binding polypeptide areminimized and/or outweighed by the beneficial effects.

As used herein, the term “subject” includes any human or non-humananimal.

As used herein, the term “surface plasmon resonance” refers to anoptical phenomenon that allows for the analysis of real-timeinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore™ system(Biacore Life Sciences division of GE Healthcare, Piscataway, N.J.).

As used herein, the term “K_(D)” refers to the equilibrium dissociationconstant of a particular binding polypeptide/antigen interaction.

As used herein, the term “off-rate” is refers to the dissociation rate(K_(off)) for a particular binding polypeptide/antigen interaction.

As used herein, the term “epitope” refers to an antigenic determinantthat interacts with the specific antigen binding site in a bindingmolecule of the invention. A single antigen may have more than oneepitope. Thus, different antibodies may bind to different areas on anantigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain.

II. BINDING POLYPEPTIDES

In one aspect, the invention provides binding polypeptides comprising aVH domain, wherein, as an isolated domain, the VH domain binds to anantigen with a Kd of less than about 200 pM (e.g., about 200, 190, 180,175, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 80, 75, 70, 65, 60,55, 50, 40, 30, 20, 10, 5, or 1 pM or less).

In another aspect, the invention provides binding polypeptides (e.g.,antibodies, or antigen binding fragments thereof) that specifically bindto PDGFRβ and inhibit PDGFRβ activity. Such binding polypeptides areparticularly useful for treating PDGFRβ-associated disease or disorders(e.g., age-related macular degeneration or AMD).

In general, PDGFRβ binding polypeptides of the invention comprise aheavy chain CDR3 (HCDR3) amino acid sequence that specifically binds toPDGFRβ. One, non-limiting, HCDR3 sequence suitable for use in thebinding polypeptides of the invention is the heavy chain CDR3 amino acidsequence set forth in SEQ ID NO: 1 (HGGDRSY)). In other embodiments, theheavy chain CDR3 sequence is a variant of SEQ ID NO:1 which comprises atleast one (e.g., one, two, or three) conservative amino acidsubstitutions relative to SEQ ID NO:1.

Any binding polypeptide that can incorporate a heavy chain CDR3 aminoacid sequence that specifically binds to PDGFRβ (e.g., the CDR3 aminoacid sequence set forth in SEQ ID NO: 1 (HGGDRSY)) can be used in thebinding polypeptides of the invention including, without limitationantibodies, or fragments thereof, and immunoglobulin-like domains.Suitable immunoglobulin-like domains include, without limitation,fibronectin domains (see, for example, Koide et al. (2007), Methods Mol.Biol. 352: 95-109, which is incorporated by reference herein in itsentirety), DARPin (see, for example, Stumpp et al. (2008) Drug Discov.Today 13 (15-16): 695-701, which is incorporated by reference herein inits entirety), Z domains of protein A (see, Nygren et al. (2008) FEBS J.275 (11): 2668-76, which is incorporated by reference herein in itsentirety), Lipocalins (see, for example, Skerra et al. (2008) FEBS J.275 (11): 2677-83, which is incorporated by reference herein in itsentirety), Affilins (see, for example, Ebersbach et al. (2007) J Mol.Biol. 372 (1): 172-85, which is incorporated by reference herein in itsentirety), Affitins (see, for example, Krehenbrink et al. (2008). J Mol.Biol. 383 (5): 1058-68, which is incorporated by reference herein in itsentirety), Avimers (see, for example, Silverman et al. (2005) Nat.Biotechnol. 23 (12): 1556-61, which is incorporated by reference hereinin its entirety), Fynomers, (see, for example, Grabulovski et al. (2007)J Biol Chem 282 (5): 3196-3204, which is incorporated by referenceherein in its entirety), and Kunitz domain peptides (see, for example,Nixon et al. (2006) Curr Opin Drug Discov Devel 9 (2): 261-8, which isincorporated by reference herein in its entirety).

In a preferred embodiment, the PDGFRβ binding polypeptides areantibodies, or antigen binding fragment thereof, comprising a VH domainand/or a VL domain. Exemplary CDR, VH, and VL amino acid sequencessuitable for use in the invention are set forth in Tables 1-4.Accordingly, in certain embodiments, the binding polypeptides maycomprise HCDR3 (SEQ ID NO:1) together with HCDR2 and/or HCDR1 sequenceswhich are independently selected from any one of the heavy chain HCDR2or HCDR1 sequences set forth in Table 1. In certain embodiments, thebinding polypeptides of the invention may further comprise light chainCDRs which are independently selected from any one of the light chainCDR1, CDR2 or CDR3 sequences set forth in Table 2. For example, thebinding polypeptide of the invention may comprise any one of the heavychain variable (VH) domains set forth in Table 3, optionally paired withany one of the light chain variable (VL) domains set forth in Table 4.

TABLE 1 Heavy chain CDR amino acid sequences of exemplary anti-PDGFRβantibodies. Clone SEQ ID SEQ ID SEQ ID name CDR3 NO. CDR2 NO. CDR1 NO.XB1511 HGGDRSY 1 GIIPIFGTANYAQKFQG 2 SYAIS 33 G2 HGGDRSY 1GIIPIFGTANYAQKFQG 2 GYAIS 34 XB2202 HGGDRSY 1 GILPILKTPNYAQRFQG 3 RHAIS35 C05. HGGDRSY 1 GVLPILKTPNYAQRFQG 4 RHAIS 35 E2. HGGDRSY 1WINPNSGNIGYAQKFQG 5 DYYIQ 36 A3. HGGDRSY 1 WINPNSGGTYFAQKFQG 6 DYYIQ 36C3. HGGDRSY 1 GILPILKTPNYAQRFQG 3 DYYIQ 36 F10. HGGDRSY 1WINPDSGGTYFAQKFQG 7 DYYIQ 36 C12. HGGDRSY 1 WMNPDSGGTIYAQKFQG 8 DYYIQ 36H2. HGGDRSY 1 WLNPNSGDTHSAQKFQG 9 AYYIQ 37 B1. HGGDRSY 1WINPNNGNITYAQKFQG 10 DYYIH 38 E11. HGGDRSY 1 GIIPIFGTANYAQKFQG 2 DYYIH38 H1. HGGDRSY 1 WINPNSGGINSAPKFQG 11 DYHLH 39 E6. HGGDRSY 1WINPNSGGTNYAQKFQG 12 DYYLH 40 A1. HGGDRSY 1 WIVVGSGNTNYAQKFQE 13 SSAVQ41 H7. HGGDRSY 1 WIVVGSGNTNYAQKFQE 13 SSAMQ 42 G04. HGGDRSY 1WIVVGSGNTNYAQKFQE 13 SYAIS 33 B2. HGGDRSY 1 VINTGVGSTNYAQKFQG 14 NYQVQ43 A7. HGGDRSY 1 VINTGVGSTNYAQKFQG 14 NYPVQ 44 H3. HGGDRSY 1LSNPSGDYTVYAPKFQG 15 NSFMQ 45 B4. HGGDRSY 1 LSNPSGDYTVYAPKLQG 16 NSFMQ45 D06. HGGDRSY 1 VISYDGSNKYYADSVKG 17 SYGMH 46 F3. HGGDRSY 1WISADNGNTNYAQKFQE 18 SHGMS 47 A12. HGGDRSY 1 WISADNGNTKYAQKFQD 19 SHGMS47 G3. HGGDRSY 1 GFDPEDGETIYAQKFQG 20 ELSMH 48 H12. HGGDRSY 1GIIPIFGTANYAQKFQG 2 DNYVH 49 G12. HGGDRSY 1 GIIPVSGTPNYAQKFQG 21 AYPIS50 C06. HGGDRSY 1 GIIPIFGTANYAQKFQG 2 GHYIH 51 C11. HGGDRSY 1GIIPIFGTANYAQKFQG 2 NDYIH 52 F08. HGGDRSY 1 GIIPIFGTANYAQKFQG 2 SSYIH 53E9. HGGDRSY 1 ITYPADSTTVYSPSFQG 22 NYWIG 54 E11. HGGDRSY 1RINNDGSSTSYADSVKG 23 SYWMH 55 C08. HGGDRSY 1 RISIDGITTTYADSVQG 24 AFWMH56 XB2708 HGGDRSY 1 FILFDGNNKYYADSVKG 25 SYGMH 46 D03. HGGDRSY 1RINADGTSTAYAESVKG 26 NDWMH 57 A10. HGGDRSY 1 LIYSDGSTYYADSVKG 27 DYAMN58 C09. HGGDRSY 1 AIDGSGGTTYYAGSVKG 28 NNAMS 59 A06. HGGDRSY 1HISNDGSITRYADSVKG 29 GHWMH 60 C05. HGGDRSY 1 RIKTDGSSTSYADSVKG 30 SNWMH61 H01. HGGDRSY 1 RISSDGSTTAYADSVRG 31 SDWMH 62 G07. HGGDRSY 1RISSDGSSTAYADSVKG 32 SDWMH 62

TABLE 2 Light chain CDR amino acid sequences of exemplary anti-PDGFRβantibodies. Clone SEQ ID SEQ ID SEQ ID name CDR3 NO. CDR2 NO. CDR1 NO.B10. HQSSSLPWT 63 AYQSVS 148 RASQTIGSTLH 233 H10. HQSSSLPHT 64 SSQSFS149 RASQSIGSSLH 234 F10. RASQSIGSGLH 65 ASQSMS 150 HQSSSLPWT 235 B12.HQTSSLPLT 66 ASQPFS 151 RASQSIGIKLH 236 B11. QQYGSSPWT 67 GASSRAS 152RASQSVSSNYLA 237 E7. QQYGSSPQT 68 GASSRAT 153 RASQSVSSSYLA 238 E8.QQYGSSPPYT 69 GASSRAT 154 RASQSVSSSYLA 239 H8. QQYAGSPFT 70 GASSRAT 155RASQSVSSNYLA 240 H12. QQFGSSPWT 71 GASRRAT 156 RASQSVRSSYVA 241 F8.QQYGSSPLT 72 VASRRVT 157 SGGRSNIGGNAVN 142 D11. QQYGASPRT 73 GASSRAT 158RASQNITSNFFA 243 G8. QQYGSALLT 74 DASNRAA 159 RASQSLSGTYLA 244 H9.QQYGNSWT 75 RASTRAT 160 RASEDIYNNYLA 245 H11. HQSRNLPFT 76 ASQSFS 161RASQSIGSSLH 246 G12. HQSRSFPLT 77 SSQSIS 162 RASESIGTALH 247 Ell.QQYETSWT 78 RASTRAT 163 RTSQILHSQYLA 248 F12. RDGLNHLV 79 GENNRPS 164QGDTLRTCYAS 249 C8. GTWDSSLSVVI 80 YDNYQRFS 165 SGSTSNIGKNFVS 250 A8.HQTGSFPYT 81 LASQSFS 166 RASRYIGSNLH 251 B8. LLSYSGPRVV 82 DTSNKQS 167GSSTGAVTSGHSPF 252 F7. QQSYRTPFS 83 WASTRES 168 KSSXSLLYRSNNKNYL 253 AB7. QVWDSSSVI 84 RDSNRPS 169 GGANIANKNVH 254 G9. KSRDSSAMRWV 85 GKDNRPS170 QGDSLRTYYAS 255 A9. LLYFNPTRV 86 DTHNRHS 171 GSSTGAVTSGHYPY 256 A11.GADHGRV 87 GIVGSKGD 172 TLSSGYSNYKVD 257 E12. QVWHSGVI 88 FDSDRPS 173GGNNIGSKSVH 258 H7. HQSRSSHT 89 YASQSFS 174 RASQNIGNSLH 259 A10.QSFDVYSHEVV 90 GNNQRPS 175 TRCTGNIASHFVQ 260 C11. MQSTHFPFT 91 EVSKRFS176 KSSQSLLNSDDGKTYL 261 Y D10. QQYDSPPWT 92 DASHLEA 177 QASHDISNYLN 262D12. QQHDTSQWT 93 GASSRAA 178 RASQSVSRTYLA 263 C7. MQGLHIPHT 94 EVSGRFS179 KSSQSLLHSDGKTHLF 264 D7. MQSTHQWT 95 SVSKRDS 180 RSSHSLVHSDGNIYLN265 C9. QQYDSYSRT 96 EASRLES 181 RASQSISSWLA 266 C12. QQSFSMRT 97GASGLQS 182 RTSQGIRNYLS 267 D8. QQYVNSRT 98 DASNRAT 183 RASQSVTSNYLA 268D9. QQYNDFFT 99 GASTRAT 184 RASQSVSSKLA 269 G7. MQATQFPS 100 KISNRMS 185RSSESPVHSDGNIYLS 270 G11. QQYGDSVFT 101 GGSIRAS 186 RASQSVSSRNLA 271 F9.QQSYSTPRT 102 AASTLHY 187 RASDNIGNYLN 272 E9. QESYSTLLYT 103 AASRLQS 188RASESISNYLN 273 B1. QVWESGSEHYV 104 DDSDRPS 189 GGNNIGYDSVH 274 E6.QVWESTSDHPT 105 YDNDRPS 190 GGNNIGATTV 275 F3. QVWDSSSDHWV 106 YDSDRPS191 GGNNIGSKSVH 276 H4. QVWDSSSGHRGV 107 DDSDRPS 192 GGNNIVSKGVH 277 H5.QVWDSATDHVV 108 SDRDRPS 193 GGNNLGSKIVH 278 B5. QVWDSDRHHVV 109 DDYGRPS194 AGNNIGGKSVQ 279 G6. QVWDINDDYAV 110 QDTKRPS 195 SGDNLGHTNAC 280 C1.QQYVSSPPMYT 111 GASSRAT 196 TASQSVSSTYLT 281 F1. QQYVTYPLT 112 GASNLEG197 RASQNIDYDLA 282 A3. QQYDSVPLT 113 GASTLES 198 QASQVIDKYVN 283 B4.QQYEDLPSF 114 EASNLET 199 QASQDIFHYLN 284 B6. QQYGSFPYS 115 AASNRAT 200RASQSFGSNYLA 285 F2. QQYQNPPFT 116 GASNLER 201 QASQFIHIYLN 286 D3.QQYKTFPHT 117 AASYLQT 202 RASQDVGIYVA 287 G2. QQYHSYPYT 118 KVSTLES 203RASQDINTWLA 288 A4. QQYNNVLRT 119 EASNLET 204 QASQDISNWLN 289 G4.QQYNKWPTF 120 GASTRAT 205 RVSQNVFSDLA 290 D5. QQYYNWPPWT 121 AASTLHY 206RASDNIGNYLN 291 A1. QQRSNGVTF 122 EASTRAT 207 RASQSVSSFLA 292 H2.QHYHTYPFT 123 QASSLKT 208 RATESISIWLA 293 E2. QQYYLTPTFTVT 124 WASTRES209 KSSQSVLYSSNNKNYL 294 A F4. QQTNTFPLT 125 RATNLQS 210 RASQDISSWLA 295C5. QQYHTTPYT 126 WASTRES 211 KSSQSVLYSSNNRNYL 296 A E5. QQSFSSPWT 127AASNLQS 212 RASQTFTSHLN 297 F6. QQSFTTLVT 128 SASTLQS 213 RASQSVNVYLN298 G5. CQQFNSYPLS 129 DASTLQT 214 RASQDISSSLA 299 A5. GADHGSGSNLVYV 130VGTGGIVGSRGD 215 TVSSGYRSYEVD 300 D6. GADHGSGSDFVYV 131 VGTGGIVGSRGD 216TLSSDYSSYNVD 301 E4. AAWDDSLNGPV 132 TNNQRPS 217 SGSSTNIGSNAVN 302 F5.AAWDDRLSGPV 133 TTDRRPS 218 SGGGSNIGSNFGY 303 G1. ATWDDDLSNPKWV 134TTNQRPS 219 SGSSSNIGSNSVD 304 E3. MQALQTSWT 135 LGSNRAS 220RSSQSLLHSNGYNFLD 305 A2. MQGTHWPYT 136 QVSTRDS 221 CDDTVSTLPARHP 306 D1.MQSRNLPKT 137 EASSRFS 222 KSSQSLVHRDGKTYLY 307 C4. MVWYSAWV 138RSDSDRHQGS 223 TLSSGFNVVSYNIY 308 E1. HVLDSSTIVI 139 RDTNRPS 224AGNNIGTYYVH 309 A6. HQYNNWPLYT 140 GASTRAT 225 RASQSVSSNLA 310 H1.NSRDSSGYLLL 141 GKNTRPS 226 QGDSLRTYYAS 311 B2. LLSYSGAGV 142 DASNKHS227 GSSTGAVTSGHYPY 312 C2. LQDYSFPYT 143 DSSTLQS 228 RPSQDIGTDLG 313 G3.QAWDSSHAV 144 QDTKRPS 229 SGDELKYKYTC 314 H3. QSEDSRGPV 145 KDTERPS 230SGSTFPKLYSF 315 D4. VQATHFPVT 146 KISNRFS 231 RSSESVVHDDGNTYLS 316 C6.CSYTTGSTLYL 147 DVNRRPS 232 TGTSDDVGRYDYVS 317

TABLE 3Heavy chain variable domain (VH) amino acid sequences of exemplary anti-PDGFRβ antibodies. Clone SEQ ID name VH Amino Acid Sequence NO.Sequences of VH Primary Selection on recombinant human PDGFRβ A4QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMG 233 XB1511GIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAI HGGDRSYWGQGTLVTVSS B4QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAISWVRQAPGQGLEWMG 234GIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAI HGGDRSYWGQGTLVTVSS G2QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYAISWVRQAPGQGLEWMG 235GIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSKDTAVYYCAI HGGDRSYWGQGTLVTVSS(XB1511) Framework Shuffled and selected with 2 rounds on human and2 rounds on mouse PDGFRβ targets XB2202QVQLVQSGAEVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQGLEWIG 236GILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSS C4.QMQLVQSGAEVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQGLEWIG 237GILPILKTPNYAQRFQGRVTINADESTSTVYMEMSGLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSSB12. QMQLVQSGAEVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQGLEWIG 238GILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSENTAVYYCAT HGGDRSYWGQGTLVTVSSD07. QMQLVQSGAEVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQGLEWIG 239GILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSDDTAVYYCAT HGGDRSYWGQGTLVTVSSC05. QMQLVQSGAEVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQGLEWIG 240GVLPILKTPNYAQRFQGRVTINADESTSTVYMELSSLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSSE05. QVQLVQSGPKVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQGLEWIG 241GILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSS E2.QMQLVQSGAEVKKPGASVKISCKTSGYTFTDYYIQWVRQAPGQGLEWVG 242WINPNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRSEDTAVYYCAT HGGDYSYWGQGTLVTVSS A3.QVQLVQSGAEVKKPGASVRVSCKASGYTFSDYYIQWVRQAPGQGLEWMG 243WINPNSGGTYFAQKFQGRVTMTRDTSISTAYMELSSLTSDDTAVYYCAT HGGDRGYWGQGTLVTVSS C3.QMQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIQWVRQAPGQGLEWIG 244GILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSSF10. QMQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIQWVRQAPGQGLEWMG 245WINPDSGGTYFAQKFQGRVAMTRDTSINTAYMELSSLRSDDTAVYYCAT HGGDRSYWGQGTLVTVSSC12. QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIQWVRQAPGEGLEWMG 246WMNPDSGGTIYAQKFQGRVTMTRDTSISTAYMELSRLRPDDTAVYYCAT HGGDRSYWGQGTLVTVSS H2.QMQLVQSGAEVKNPGASVKVSCKASGYPFSAYYIQWVRQAPGQGLEWMG 247WLNPNSGDTHSAQKFQGRVTMTRDTSISTAYMELSGLTSDDTAVYYCAT HGGDRSYWGQGTLVTVSSF11. QMQLVQSGAEVKNPGASVKVSCKASGYPFSAYYIQWVRQAPGQGLEWMG 248WLNPNSGDTHSAQKFQGRVTMTRDTSISTAYMELSGPTSDDTAVYYCAT HGGDRSYWGQGTLVTVSS B1.QMQLVQSGAEVRKPGASVKVSCKASGYSFSDYYIHWVRQAPGQGLEWIG 249WINPNNGNTTYAQKFQGRVTMIRDTSISTAYMELSELRSDDTAVYYCAT HGGDRSYWGQGTLVTVSSE11. QVQLVQSGAEVEKPGASVKVSCKASGYTFTDYYIHWVRQAPGQGLEWMR 250GIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSS H1.EVQLLESGAEVKQPGASVKVSCKTSGYTFTDYHLHWVRQAPGQGLEWMG 251WINPNSGGTNSAPKFQGRVTMTRDTSISTAYMELSGLTSDDTAVYYCAT HGGDRSYWGQGTLVTVSS E6.QMQLVQSGAEVKRPGASVKVPCKASGYTFTDYYLHWVRQAPGQGLKWMG 252WINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSS A1.QVQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAVQWVRQARGQRLEWIG 253WIVVGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSS H7.QVQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAMQWVRQARGQRLEWIG 254WIVVGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSSG04. QVQLVQSGAEVKKPGASVKVSCKASGFTFTSYAISWVRQARGQRLEWIG 255WIVVGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSS B2.QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYQVQWVRQAPGQGLEWLG 256VINTGVGSTNYAQKFQGRVTMTRDTATSTVYMELSSLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSS A7.QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYPVQWVRQAPGQGLEWLG 257VINTGVGSTNYAQKFQGRVTMTRDTATSIVYMELSSLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSS H3.QVQLVQSGAEVKKPGASVKVSCRASGYTFTNSFMQWVRQVPGQRLEWVG 258LSNPSGDYTVYAPKFQGRVTMTTDTATSTFYMELFSLRSDDTAVYYCAT HGGDRSYWGQGTLVTVSS B4.QVQLVQSGAEVKKPGASVKVSCRASGYTFTNSFMQWVRQVPGQRLEWVG 259LSNPSGDYTVYAPKLQGRVTMTTDTATGTFYMELFSLRSDDTAVYYCAT HGGDRSYWGQGTLVTVSSH05. EVQLVQSGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQAPGKGLEWVA 260FILFDGNNKYYADSVKGRFTISSDNSKNTLYLQMNSLRAEDTAVYYCAT HGGDRSYWGQGTLVTVSSD06. QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVA 261VISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK HGGDRSYWGQGTLVTVSS F3.QVQLVQSGAEVKKPGASVKVSCKASGYTFISHGMSWVRQAPGQGLEWMG 262WISADNGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSSA12. QVQLVQSGAEVKKPGASVKVSCKASGYTFISHGMSWVRQAPGQGLEWMG 263WISADNGNTKYAQKFQDRVTLTTDTSTSTAYLELRSLRSDDTAVYYCAT HGGDRSYWGQGTLVTVSS G3.QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMG 264GFDPEDGETIYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAT HGGDRSYWGQGTLVTVSSF05. QVQLVQSGAEVKRPGASVKVSCKASGYTLTELSMHWVRQAPGKGLEWMG 265GFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSSH12. QVQLVQSGAEVKKPGASVKVSCKASGYTFTDNYVHWVRQAPGQGLEWMG 266GIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSSG12. QVQLVQSGAEVKKPGSSVKVSCKASGGAFNAYPISWVRQAPGQGLEWMG 267GIIPVSGTPNYAQKFQGRVTITADKSTYTAYMELSSLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSSC06. QMQLVQSGAEVKKPGASVKVSCMASGYTFTGHYIHWVRQAPGQGLEWMG 268GIIPIFGTANYAQKFQGRVTITADESTSTAYTELSSLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSSC11. QVQLVQSGAAVKKPGASVKVSRKASGYTFTNDYIHWVRQAPGQGLEWMG 269GIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSSF08. QVQLVQSGAEVKKPGASVKVSCKASGYTFTSSYIHWVRQAPGQGLEWMG 270GIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAMYYCAT HGGDRSYWGQGTLVTVSS E9.QVQLVESGAEVRKPGESLQISCKASGYRFTNYWIGWVRQMPGKGLEWMG 271ITYPADSTTVYSPSFQGQVTISADKSISTVFLQWSSLRSEDTAVYYCAT HGGDRSYWGQGTLVTVSSE11. QVQLVESGGGVVQPGRSLRLSGAASGFTFSSYWMHWVRQAPGKGLVWVS 272RINNDGSSTSYADSVKGRFTISRDTAKNTLYLQMNSLRAEDTAVYYCAT HGGDRSYWGQGTLVTVSSH11. QVQLLESGAEVKNPGASVKVSCKASGYPFSAYYIQWVRQAPGQGLEWMG 273WLNPNSGDTHSAQKFQGRVTMTRDTSISTAYMELSGLTSDDTAVYYCAT HGGDRSYWGQGTLVTVSSC08. EVQLLESEGGLVQPGGSLRLSCTASGFSFNAFWMHWVRQAPGKGLEWVS 274RISIDGTTTTYADSVQGRFTISRDNARNTLYLQMNSLRAEDAAVYYCAT HGGDRSYWSQGTLVTVSS(XB1511) Framework Shuffled and selected with human PDGFRβ andoff rate selection XB2708QVQLVQSGGGVVQPGGSLRLSCAASGFTSRSYGMHWVRQAPGKGLEWVA 275FILFDGNNKYYADSVKGRFTISSDNSKNTLYLQMNSLRAEDTAVYYCAT HGGDRSYWGQGTLVTVSSD03. QVQLVQSGGGLVQPGGSLRLSCVASGFTFGNDWMHWVRQAPGKGLVWVS 276RINADGTSTAYAESVKGRFTVSRDNAKNTLYLQMNGLRAEDTAVYYCAT HGGDRSYWGQGTLVTVSSA10. QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMNWVRQAPGKGLEWVS 277LIYSDGSTYYADSVKGRFTISRDNSKKTLYLQMNNLRVEDTAVYYCATH GGDRSYWGQGTLVTVSS C09.QVQLVQSGGALVQPGGSLRLSCAASGFTLSNNAMSWVRQAPGKRLEWVS 278AIDGSGGTTYYAGSVKGRFTISSDNSKNTVFLQMNSLRAEDTAVYYCAT HGGDRSYWGQGTLVTVSSA06. QVQLVQSGGGLVQPGGSLRLSGAASGFTFSGHWMHWVRQVPGKGLVWVS 279HISNDGSITRYADSVKGRFTVARDNAKNTMYLQMNSLRAEDTAVYYCAT HGGDRSYWGQGTLVTVSSC05. QVQLVQSGGGLVKPGGSLRLSCAASGFIFSSNWMHWVRQVPGKGLEWVS 280RIKTDGSSTSYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT HGGDRSYWGQGTLVTVSSH01. QVQLVQSGGGLVQPGGSLRLSGAASGFTLSSDWMHWVRQAPGKGLVWVS 281RISSDGSTTAYADSVRGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT HGGDRSYWGQGTLVTVSSG04. QVQLVQSGGGLVQPGGSLRLSGAASGFTLSSDWMHWVRQAPGKGLVWVS 282RISSDGSTTAYADSVRGRFTISRDNTKNTLYLQMNSLRAEDTAVYYCAT HGGDRSYWGQGTLVTVSSG07. QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSDWMHWVRQAPGEGLVWVS 283RISSDGSSTAYADSVKGRFTISRDNAKNTVSLQMNSLRAEDTAVYYCAT HGGDRSYWGQGTLVTVSS

TABLE 4Light chain variable domain (VL) amino acid sequences of exemplary anti-PDGFRβ antibodies. Clone SEQ ID name VL Amino Acid Sequence NO.PR2 VL sequences from XB1511 pairing B10.QSVLTQSPDLQSVTPREKLTITCRASQTIGSTLHWYQQKPGQSPRLVIKY 284AYQSVSGVPSRFSGSGSGTEFTLTINGLEAEDAATYYCHQSSSLPWTFGQ GTKLTVL H10.QSVLTQSPDFQSVSPKDKVTITCRASQSIGSSLHWYQQKPGQSPKLLIKY 285SSQSFSGVPSRFSGSASGTEFTLTITGLEAEDAATYYCHQSSSLPHTFGQ GTKVTVL F10.QSVLTQSPEFQSVTPKEKVTITCRASQSIGSGLHWYQQKPHQSPKLLIRY 286ASQSMSGVPSRFSGSGSGTDFTLTISRLEVEDAAMYYCHQSSSLPWTFGQ GTKVTVL B12.QSVLTQSPDFQSVTPKQNVTFTCRASQSIGIKLHWYQQKPDQSPKVLIKY 287ASQPFSGVPSRFSGRGSGTDFTLTINSLEAEDAATYYCHQTSSLPLTFGG GTKVTVL B11.QSVLTQSPGTLSLSPGERATLSCRASQSVSSNYLAWYQQKPGQAPRLLIY 288GASSRASGIPVRVSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFG QGTKLTVL E7.QSVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIY 289GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPQTFG QGTKLTVL E8.QSVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIY 290GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPYTF GQGTKLTVL H8.QSVLTQSPGTLSLSPGERATLSCRASQSVSSNYLAWYLQKPGQAPRLLIS 291GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYAGSPFTFG PGTKLTVL H12.QSVLTQSPGTLSLSPGERATLSCRASQSVRSSYVAWYQQKPGQAPRLLIS 292GASRRATGIPDRFTGSGSGTDFTLTISRLEPEDFAVYHCQQFGSSPWTFG QGTKLTVL F8.QSVLTQPPSASGTPGQRVTISCSGGRSNIGGNAVNWYQQKPGQAPRLLTH 293VASRRVTGIPDRFSGSGSGTDFTLTISRLEPEDFAIYYCQQYGSSPLTFG GGTKLTVL D11.QSVLTQSPGTLSLSPGERATLSCRASQNITSNFFAWYQQKPGQAPRLLIY 294GASSRATGIPDRISGSGSGTDFTLTISRLEPEDFALYYCQQYGASPRTFG QGTQLTVL G8.QSVLTQSPGTLSLSPGDRATLSCRASQSLSGTYLAWYQQKPGQAPRLLIY 295DASNRAAGIPKRFSGSGSRTDFTLTISRVDPADSAVYYCQQYGSALLTFG GGTKVTVL H9.QSVLTQSPGTLSLSPGESATLSCRASEDLYNNYLAWYQHKRGQPPRLLIF 296RASTRATGIPTRFSGSGSGRDFVLTINRLEPEDFAVYYCQQYGNSWTFGQ GTKLTVL H11.QSVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPDQSPKLLITF 297ASQSFSGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHQSRNLPFTFGP GTKLTVL G12.QSVLTQSPDFQSVTPKEEVTITCRASESIGTALHWYQQKPDQSPKLLIKY 298SSQSISGVPSRFVGRGSETEFTLTINSLEAENAATYYCHQSRSFPLTFGQ GTQLTVL E11.QSVLTQSPGTLSLSPGERATLSCRTSQILHSQYLAWYQQKRGQAPRLLIF 299RASTRATGIPERFSGSGSGRDFVLTISRLEPEDSAVYYCQQYETSWTFGQ GTKVTVL F12.QSVLTQDPVVSVALGQTVRITCQGDTLRTCYASWYQQRPRQAPILVIYGE 300NNRPSGIPARFSGSSSGSTASLTITGAQAEDEGDYYCHCRDGLNHLVFGG GTKVTVL C8.QSVLTQPPSVSAAPGQKVTISCSGSTSNIGKNFVSWYQHLPGTAPKLLIY 301DNYQRFSGIPDRFSGFKSGTSATLSITGLQTADEADYYCGTWDSSLSVVI FGGGTKLTVL A8.QAGLTQSPDFQSVTPKERVTITCRASRYIGSNLHWYQQKPDQPPKLLIKL 302ASQSFSGVPPRFSGGGSGTDFTLTINGLEAEDAATYYCHQTGSFPYTFGQ GTKLTVL B8.QAVLTQEPSLTVSPGGTVTLTCGSSTGAVTSGHSPFWFQQRPGQAPRTLI 303YDTSNKQSWTPARFSGSLLGGKAALTLSGAQPEDEAEYYCLLSYSGPRVV FGGGTKVTVL F7.QAVVTQSPDSLAVSLGERATISCKSSXSLLYRSNNKNYLAWYQQKPGQPP 304RLLISWASTRESGVPDRFSGSGSGTDFTLTVSRLRAEDAAVYYCQQSYRT PFSFGPGTKVTVL B7.SYVLTQPLSVSVALGQTARISCGGANIANKNVHWYQLQPGQAPVLVIYRD 305SNRPSGIPERFSGSNSGNTATLTITRAQARDEADYYCQVWDSSSVIIGGG TKVTVL G9.SYVLTQDPAVSVALGQTVRITCQGDSLRTYYASWYRQKPGQAPVLVFYGK 306DNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCKSRDSSAMRWVFG GGTKLTVL A9.NFMLTQEPSLTVSPGGTVTLTCGSSTGAVTSGHYPYWFQQKPGQVPRTFI 307YDTHNRHSWTPVRFSGSLFGGKAALTLSGAQPEDEAEYYCLLYFNPTRVF GGGTKLTV A11.NFMLTQPPSASASLGASVTLTCTLSSGYSNYKVDWYQQRPGKGPRFVMRV 308GTGGIVGSKGDGIPDRFSVLGSGLNRYLTIKNIQEEDESDYHCGADHGRV FGGGTKLTVL E12.QPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYHLRPGQAPVLVIYFD 309SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWHSGVIFGGGT KLTVL H7.QPVLTQSLDFQSVTPKEKVTITCRASQNIGNSLHWYQQKPNQSPKVLIKY 310ASQSFSGVPSRFSGSGFGTDFTLTINSLEPEDAATYYCHQSRSSHTFGQG TKLTVL A10.EIVLTQSPGNLSLSPGERATLSCTRCTGNIASHFVQWYQQRPGSSPTTVI 311FGNNQRPSGVSDRFSGSIDSSSNSASLTISRLKTEDEADYYCQSFDVYSH EVVFGGGTKLTVL C11.QTVVTQTPVSLSVTPGQPASISCKSSQSLLNSDDGKTYLYWYLQRPGQPP 312HLLIYEVSKRFSGVPDRFSGSGSGTDFTLRISRVEAEDVGVFYCMQSTHF PFTFGPGTKVTVL D10.NIQMTQSPVSLSASLGDTVSITCQASHDISNYLNWYQQKPGKAPKLLIYD 313ASHLEAGVPSRFRGSGSGTDFTLTINRLEPEDFAVYYCQQYDSPPWTFGQ GTKLTVL D12.DVVLTQSPGTMSLSPGERATLSCRASQSVSRTYLAWHQQKPGQAPRLLIY 314GASSRAAGIPDRFSGSGSGTDFTLSISRLEPEDFAVYYCQQHDTSQWTFG QGTKLTVL C7.DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTHLFWYLQRPGQSPQ 315LLIYEVSGRFSGVSERFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLHIP HTFGQGTKVEIK D7.DIVMTQSPLSLPVTLGQPASISCRSSHSLVHSDGNIYLNWYHQRPGQSPR 316RLIYSVSKRDSGVPDRFSGSGSRTDFTLKISRVEAEDVGVYFCMQSTHQW TFGQGTKVEIK C9.VIWMTQSPSTVSASVGDRVTITCRASQSISSWLAWYQQKPGKAPNLLIYE 317ASRLESGIPSRFSGSGSGTEFTLTXSSLQPDDFATYYCQQYDSYSRTFGQ GTKVAIK C12.DVVMTQSPSSLSASVGDRVTITCRTSQGIRNYLSWYQQKPAKAPKLLIHG 318ASGLQSGVPSRFSGSGSGTNFTLTISSLQPEDFATYYCQQSFSMRTFGQG TKVEIK D8.EIVMTQSPGTLTLSPGEGATLSCRASQSVTSNYLAWYQQRPGASSLQSGQ 319APRLLIYDASNRATGIPDRFSGSGFGTDFTLTISRLEPEDFAVYYCQQYV NSRTFGQGTKVEIK D9.EIVMTQSPVTLSVSPGERATLSCRASQSVSSKLAWYQQKPGQAPRLLIYG 320ASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAIYYCQQYNDFFTFGPG TKVDIK G7.EIVLTQTPLSSPVTLGQPASISCRSSESPVHSDGNIYLSWLHQRPGQPPR 321LLLYKISNRMSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFP SFGQGTKLEIK G11.EIVLTQSPGTLSLSPGEGATLSCRASQSVSSRNLAWYQQKPGQAPRLLIY 322GGSIRASGTSTRFSGSGSGTDFTLTINRLEPEDFAVYYCQQYGDSVFTFG PGTKVDIK F9.NIQMTQSPSSLSASVGDRVNITCRASDNIGNYLNWYQHKPGKAPTVLIYA 323ASTLHYGVPSRFSGRGSGTDFTVTISSLQPEDSATYYCQQSYSTPRTFGQ GTRVELK E9.AIQMTQSPSSLSASVGDRVTITCRASESISNYLNWYQQKPGKAPKLLLSA 324ASRLQSGVPSRFSGSGSGTDFTLTITSLQPEDLATYYCQESYSTLLYTFG QGTKLEIKVL sequences from XB2202 VL pairing B1.SYELTQPPSVSVAPGKTASITCGGNNIGYDSVHWYQQKPGQAPVLVVFDD 325SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWESGSEHYVFG TGTQLTVL E6.LPVLTQPPSVSVAPGQTARISCGGNNIGATTVHWYQHRPGQAPVSVIFYD 326NDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWESTSDHPTFG GGTQLTVL F3.QSVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYD 327SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHWVFG GGTKLTVL H4.SYELTQSPSVSVPPGQTARITCGGNNIVSKGVHWYQQRPGQAPVLVVYDD 328SDRPSGIPERFAGFNSGNTATLTISRVEAGDEADYYCQVWDSSSGHRGVF GGGTKVTVL H5.SYELTQPPSVSMAPGKTARITCGGNNLGSKIVHWYQQKPGQAPVVVIYSD 329RDRPSGVPERFSGSNSGNSATLTISGVEAGDEADYYCQVWDSATDHVVFG GGTKLTVL B5.SYELTQPPSVSVAPGQTATITCAGNNIGGKSVQWYQQKPGQAPVVVVYDD 330YGRPSGIPERVSGSNSGNTATLTLTRVEAGDEADYYCQVWDSDRHHVVFG GGTKLTVL G6.QLVLTQPPSVSVSPGQTASITCSGDNLGHTNACWYQQNPGQSPVLVIYQD 331TKRPSGIPERFSGSNSGNPATLTIXRVXAGDEANYYCQVWDINDDYAVFG TGTXLTVL C1.QSVLTQSPGTLSLSPGERATLSCTASQSVSSTYLTWYQQKPGQAPRLLIY 332GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYVSSPPMYT FGQL F1.DIQMTQSPSTLSASVGDRVTISCRASQNIDYDLAWYQXKPGKAPXLLIYG 333ASNLEGGVPSXFSGXGSGTEFTLTISSLQPDXSATYYCQQYVTYPLTFGQ GTRLEIK A3.AIQMTQSPSSLSASVGDRVTMTCQASQVIDKYVNWYRQRPGKAPELLIYG 334ASTLESGVPSRFSGSGSGTQFTFSITSVQPEDFATYICQQYDSVPLTFGP GTILDVKRTVA B4.DIQLTQSPSSLSASIGDRVTITCQASQDIFHYLNWFQQKPGKAPKLLIYE 335ASNLETGVPSRFSGSGSVTDFTFTISSLQPEDIATYYCQQYEDLPSFGGG TKVDIKRTVA B6.EIVLTQSPGTLSLSPGERATLSCRASQSFGSNYLAWYQHKPGQAPRLLIF 336AASNRATGIPDRFTGSASGTDFTLTINRVEPEDLAVYYCQQYGSFPYSFG QGTKLEIK F2.NIQMTQSPSSLSASVGDRVTITCQASQFIHIYLNWYQQKLGKAPKLLIYG 337ASNLERGVPSRFSGRGSETDFTFTIDSLQPEDIATYFCQQYQNPPFTFGG GTKVEINGTVA D3.AIRMTQSPSSLSASIGDRISVTCRASQDVGIYVAWFQQKPGKPPRSLIYA 338ASYLQTAVPPKFRGSGSGTDFTLTISDLQPDDFATYYCQQYKTFPHTFGQ GTKLDFKRTVA G2.VIWMTQSPSTLSASVGDRVTITCRASQDINTWLAWYQQKPGKAPKLLMFK 339VSTLESGDFSRFSGSGSGTEFTLTVSSLQPDDSAIYYCQQYHSYPYTFGQ GTRLEIK A4.DVVMTQSPSSLSASVGDRVTITCQASQDISNWLNWYQQKPGKAPKLLIYE 340ASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYNNVLRTFGQ GTKVEIK G4.EIVMTXSPATLSVSPGERVTLSCRVSQNVFSDLAWYQRKTGQSPRLLIHG 341ASTRATGIPTRFSGSGSGTEFTLTISSLXSDDFAVYYCQQYNKWPTFGQG TKVEIK D5.AIQLTQSPSSLSASVGDRVNITCRASDNIGNYLNWYQHKPGKAPTVLIYA 342ASTLHYGVPSRFSGRGSGTDFTVTISSLRSDDFAVYYCQQYYNWPPWTFG QGTTVDIKRTVA A1.EIVLTQSPATLSLSPGERATLSCRASQSVSSFLAWYQQKPGQAPRLLIFE 343ASTRATGISARFSGSGSGTDFTLTISTLEPEDFAVYYCQQRSNGVTFGQG TRLEIK H2.DIQMTQSPSTLSASVGDTVTITCRATESISIWLAWYQQEPGKAPNLLVSQ 344ASSLKTGVPSRFSASGSGTEFTLTISSLHPDDFATYVCQHYHTYPFTFGP GTKVDMKRTVA E2.EIVLTQSPDSXAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPP 345RLLIYWASTRESGVPDRFSGSGSGTDFTLTISRLQAEDVAVYYCQQYYLT PTFTVTFGQGTKLEIK F4.DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKFLIYR 346ATNLQSGVPSRFSGSGSGTDFTLTISSLQPGDFATYYCQQTNTFPLTFGG GTKVEVKRTVA C5.DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNRNYLAWYQKKPGQPP 347KLLFYWASTRESGVSDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYHTT PYTFGQGTKLEIK E5.VIWMTQSPSSLSASVGDRVSITCRASQTFTSHLNWYQQKPGQPPKLLIFA 348ASNLQSGVPSRFSGSGSGTDFTLTINGLQATDFATYYCQQSFSSPWTFGQ GTTVDVKGTVA F6.DIQMTQSPSSLSASVGDRVTITCRASQSVNVYLNWYQQKPGKAPKLLIYS 349ASTLQSGVPSRFTGSGSRTDFTLTINGLQPEDFATYYCQQSFTTLVTFGP GTRVDVTRTVA G5.DIQMTQSPSSLSASVGDRVTITCRASQDISSSLAWYQQKPGKAPKPLIYD 350ASTLQTGVPSRFSGRASGTDFTLTIDSLQPEDFATYCCQQFNSYPLSFGG GTKVELKRTVA A5.SYELTQPPSASASLGASVTLTCTVSSGYRSYEVDWFQQRPGKGPRFVMRV 351GTGGIVGSRGDGIPDRFSVWGSGLNRYLTIEDIQEEDESDYYCGADHGSG SNLVYVFGTGTKVTVL D6.QLVLTQPPSASASLGASVTLTCTLSSDYSSYNVDWYQQRPGMGPRFLMRV 352GTGGIVGSRGDGIPDRFSVKGSGLNRYLTIKNIQEEDESDYYCGADHGSG SDFVYVFGIGTKLTVL E4.QSVLTQPPSASGTPGQRVTISCSGSSTNIGSNAVNWYQQLPRTAPKLLIY 353TNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEANYYCAAWDDSLNGPV FGGGTQLTVL F5.QSVLTQPPSASGTPGQTVIISCSGGGSNIGSNFGYWYQQFPGTAPKLLIY 354TTDRRPSGVPDRFSGSKSGTTASLAISGLRSEDEADYYCAAWDDRLSGPV FGGGTQLTVL G1.QTVVTQPPSVSGTPGQRVTISCSGSSSNIGSNSVDWYQQFPGSAPKLLIY 355TTNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDDDLSNPK WVFGGGTKLTVL E3.DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNFLDWYLQKPGQSPQ 356LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQALQTS WTFGQGTKLEIK A2.DICRIRPLIRLTIGTITIYNYNGCCDDTVSTLPARHPWTAGLHLQSPRRL 357MYQVSTRDSGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQGTHWPYT FGQGTKLEIRRTVA D1.DIVMTQTPLSLSVTPGQPAAISCKSSQSLVHRDGKTYLYWYLQKPGHSPQ 358LLVYEASSRFSGVPDRISGSASGTQFTLNISRVEAEDVGLYYCMQSRNLP KTFGQGTKVEIK C4.SYELTQPTSLSASPGASASLTCTLSSGFNVVSYNIYWYQQKPGSPPQYLL 359RYRSDSDRHQGSGVPSRFSGSKDASANAGILVISALQSDDEADYYCMVWY SAWVFGGG E1.SYELTQPLSVSVALGQTATITCAGNNIGTYYVHWYQQRPGQAPVLVMYRD 360TNRPSGISDRFSGSNSGDTATLTICGVQVGDEADYYCHVLDSSTIVIFGG GTQLTVL A6.QSVLTQSPATLSVSPGERASLSCRASQSVSSNLAWYQQKPGQAPRLLIYG 361ASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCHQYNNWPLYTFG QGTKLTVL H1.QSVLTQDPAVPVALGQTVRITCQGDSLRTYYASWYQQKPGQAPLLVIYGK 362NTRPSGIPVRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGYLLLFG TGTKLTVL B2.QAVLTQEPSLTVSPGGTVTLTCGSSTGAVTSGHYPYWFQQKPGQAPRTLI 363YDASNKHSWTPARFSGSLLGGKAALTLSGAQPEDEAEYYCLLSYSGAGVF GTGTKVTVL C2.DIQMTQSPSSLSASVGDRVAIACRPSQDIGTDLGWYQQKPGKAPKLLIFD 364SSTLQSGVPSRFSGSLSGTDFILTITNLQPEDFATYYCLQDYSFPYTFGQ GTKLQIKRTVA G3.SYVLTQPPSVSVSPGQTASITCSGDELKYKYTCWYHQKPGQSPVLLIYQD 365TKRPSGIPERFSGSRSENTATLTISGTQAMDEADYYCQAWDSSHAVFGRG TQLTVL H3.H3SYVLTQPPSVSVFPGQTARITCSGSTFPKLYSFWYQQKTGQAPLLVIY 366KDTERPSGIPERFSGSTSGTTVTLIISGVQPEDDADYYCQSEDSRGPVFG GGTKVTVL D4.GVVMTQTPLSSLVTLGQPASISCRSSESVVHDDGNTYLSWLQQRPGQPPR 367LLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEPEDVGVYYCVQATHFP VTFGGGTRVEIK C6.QSALTQPASVSASPGQSVTISCTGTSDDVGRYDYVSWYQQHPGGAPKLIL 368YDVNRRPSGVSDRFSGSKSANKASLTISGLQADDEGDYYCCSYTTGSTLY LFGTGTQLTVL

In certain embodiments, the antibody, or antigen binding fragmentthereof, comprises a heavy chain CDR3 sequence of SEQ ID NO:1 togetherwith one or more CDR region amino acid sequences selected from the groupconsisting of SEQ ID NOs: 2-317. In exemplary embodiments, the antibody,or antigen binding fragment thereof, comprises HCDR3, HCDR2 and HCDR1amino acid sequences selected from the group consisting of SEQ ID NO: 1,2 and 3; 1, 2 and 34; 1, 3 and 35; 1, 4 and 35; 1, 5 and 36; 1, 6 and36; 1, 3 and 36; 1, 7 and 36; 1, 8 and 36; 1, 9 and 36; 1, 10 and 38; 1,2 and 38; 1, 11 and 39; 1, 12 and 40; 1, 13 and 41; 1, 13 and 42; 1, 13and 33; 1, 14 and 43; 1, 14 and 44; 1, 15 and 45; 1, 16 and 45; 1, 17and 46; 1, 18 and 47; 1, 19 and 47; 1, 20 and 48; 1, 2 and 49; 1, 21 and50; 1, 2 and 51; 1, 2 and 52; 1, 2 and 53; 1, 22 and 54; 1, 23 and 55;1, 24 and 56; 1, 25 and 46; 1, 26 and 57; 1, 27 and 58; 1, 28 and 59; 1,29 and 60; 1, 30 and 61; 1, 31 and 62; and, 1, 32 and 62, respectively.

In other embodiments, the antibody, or antigen binding fragment thereof,further comprises the LCDR3, LCDR2 and LCDR1 amino acid sequencesselected from the group consisting of SEQ ID NO: 63, 148 and 233; 64,149 and 234; 65, 150 and 235; 66, 151 and 236; 67, 152 and 237; 68, 153and 238; 69, 154 and 239; 70, 155 and 240; 71, 156 and 241; 72, 157 and242; 73, 158 and 243; 741, 159 and 244; 75, 160 and 245; 76, 161 and246; 77, 162 and 247; 78, 163 and 248; 79, 164 and 249; 80, 165 and 250;81, 166 and 251; 82, 167 and 252; 83, 168 and 253; 84, 169 and 254; 85,170 and 255; 86, 171 and 256; 87, 172 and 257; 88, 173 and 258; 89, 174and 259; 90, 175 and 260; 91, 176 and 261; 92, 177 and 262; 93, 178 and263; 94, 179 and 264; 95, 180 and 265; 96, 181 and 266; 97, 182 and 267;98, 183 and 268; 99, 184 and 269; 100, 185 and 270; 101, 186 and 271;102, 187 and 272; 103, 188 and 273; 104, 189 and 274; 105, 190 and 275;106, 191 and 276; 107, 192 and 277; 108, 193 and 278; 109, 194 and 279;110, 195 and 280; 111, 196 and 281; 112, 197 and 282; 113, 198 and 283;114, 199 and 284; 115, 200 and 285; 116, 201 and 286; 117, 202 and 287;118, 203 and 288; 119, 204 and 289; 120, 205 and 290; 121, 206 and 291;122, 207 and 292; 123, 208 and 293; 124, 209 and 294; 125, 210 and 295;126, 211 and 296; 127, 212 and 297; 128, 213 and 298; 129, 214 and 299;130, 215 and 300; 131, 216 and 301; 132, 217 and 302; 133, 218 and 303;134, 219 and 304; 135, 220 and 305; 136, 221 and 306; 137, 222 and 307;138, 223 and 308; 139, 224 and 309; 140, 225 and 310; 141, 226 and 311;142, 227 and 312; 143, 228 and 313; 144, 229 and 314; 145, 220 and 315;146, 231 and 316; and, 147, 232 and 317, respectively.

In other embodiments, the antibody, or antigen binding fragment thereof,comprises the HCDR3 amino acid sequence set forth in SEQ ID NO: 1, andLCDR3, LCDR2 and LCDR1 amino acid sequences selected from the groupconsisting of SEQ ID NO: 63, 148 and 233; 64, 149 and 234; 65, 150 and235; 66, 151 and 236; 67, 152 and 237; 68, 153 and 238; 69, 154 and 239;70, 155 and 240; 71, 156 and 241; 72, 157 and 242; 73, 158 and 243; 741,159 and 244; 75, 160 and 245; 76, 161 and 246; 77, 162 and 247; 78, 163and 248; 79, 164 and 249; 80, 165 and 250; 81, 166 and 251; 82, 167 and252; 83, 168 and 253; 84, 169 and 254; 85, 170 and 255; 86, 171 and 256;87, 172 and 257; 88, 173 and 258; 89, 174 and 259; 90, 175 and 260; 91,176 and 261; 92, 177 and 262; 93, 178 and 263; 94, 179 and 264; 95, 180and 265; 96, 181 and 266; 97, 182 and 267; 98, 183 and 268; 99, 184 and269; 100, 185 and 270; 101, 186 and 271; 102, 187 and 272; 103, 188 and273; 104, 189 and 274; 105, 190 and 275; 106, 191 and 276; 107, 192 and277; 108, 193 and 278; 109, 194 and 279; 110, 195 and 280; 111, 196 and281; 112, 197 and 282; 113, 198 and 283; 114, 199 and 284; 115, 200 and285; 116, 201 and 286; 117, 202 and 287; 118, 203 and 288; 119, 204 and289; 120, 205 and 290; 121, 206 and 291; 122, 207 and 292; 123, 208 and293; 124, 209 and 294; 125, 210 and 295; 126, 211 and 296; 127, 212 and297; 128, 213 and 298; 129, 214 and 299; 130, 215 and 300; 131, 216 and301; 132, 217 and 302; 133, 218 and 303; 134, 219 and 304; 135, 220 and305; 136, 221 and 306; 137, 222 and 307; 138, 223 and 308; 139, 224 and309; 140, 225 and 310; 141, 226 and 311; 142, 227 and 312; 143, 228 and313; 144, 229 and 314; 145, 220 and 315; 146, 231 and 316; and, 147, 232and 317, respectively.

In other embodiments, the antibody, or antigen binding fragment thereof,comprises HCDR3, HCDR2 and HCDR1 amino acid sequences selected from thegroup consisting of SEQ ID NO: 1, 2 and 3; 1, 2 and 34; 1, 3 and 35; 1,4 and 35; 1, 5 and 36; 1, 6 and 36; 1, 3 and 36; 1, 7 and 36; 1, 8 and36; 1, 9 and 36; 1, 10 and 38; 1, 2 and 38; 1, 11 and 39; 1, 12 and 40;1, 13 and 41; 1, 13 and 42; 1, 13 and 33; 1, 14 and 43; 1, 14 and 44; 1,15 and 45; 1, 16 and 45; 1, 17 and 46; 1, 18 and 47; 1, 19 and 47; 1, 20and 48; 1, 2 and 49; 1, 21 and 50; 1, 2 and 51; 1, 2 and 52; 1, 2 and53; 1, 22 and 54; 1, 23 and 55; 1, 24 and 56; 1, 25 and 46; 1, 26 and57; 1, 27 and 58; 1, 28 and 59; 1, 29 and 60; 1, 30 and 61; 1, 31 and62; and, 1, 32 and 62, respectively, and LCDR3, LCDR2 and LCDR1 aminoacid sequences selected from the group consisting of SEQ ID NO: 63, 148and 233; 64, 149 and 234; 65, 150 and 235; 66, 151 and 236; 67, 152 and237; 68, 153 and 238; 69, 154 and 239; 70, 155 and 240; 71, 156 and 241;72, 157 and 242; 73, 158 and 243; 741, 159 and 244; 75, 160 and 245; 76,161 and 246; 77, 162 and 247; 78, 163 and 248; 79, 164 and 249; 80, 165and 250; 81, 166 and 251; 82, 167 and 252; 83, 168 and 253; 84, 169 and254; 85, 170 and 255; 86, 171 and 256; 87, 172 and 257; 88, 173 and 258;89, 174 and 259; 90, 175 and 260; 91, 176 and 261; 92, 177 and 262; 93,178 and 263; 94, 179 and 264; 95, 180 and 265; 96, 181 and 266; 97, 182and 267; 98, 183 and 268; 99, 184 and 269; 100, 185 and 270; 101, 186and 271; 102, 187 and 272; 103, 188 and 273; 104, 189 and 274; 105, 190and 275; 106, 191 and 276; 107, 192 and 277; 108, 193 and 278; 109, 194and 279; 110, 195 and 280; 111, 196 and 281; 112, 197 and 282; 113, 198and 283; 114, 199 and 284; 115, 200 and 285; 116, 201 and 286; 117, 202and 287; 118, 203 and 288; 119, 204 and 289; 120, 205 and 290; 121, 206and 291; 122, 207 and 292; 123, 208 and 293; 124, 209 and 294; 125, 210and 295; 126, 211 and 296; 127, 212 and 297; 128, 213 and 298; 129, 214and 299; 130, 215 and 300; 131, 216 and 301; 132, 217 and 302; 133, 218and 303; 134, 219 and 304; 135, 220 and 305; 136, 221 and 306; 137, 222and 307; 138, 223 and 308; 139, 224 and 309; 140, 225 and 310; 141, 226and 311; 142, 227 and 312; 143, 228 and 313; 144, 229 and 314; 145, 220and 315; 146, 231 and 316; and, 147, 232 and 317, respectively.

In other embodiments, the antibody, or antigen binding fragment thereof,comprises at least one of the VH amino acid sequences set forth in SEQID NO: 318-368.

In other embodiments, the antibody, or antigen binding fragment thereof,comprises at least one of the VL amino acid sequences set forth in SEQID NO: 369-453.

In other embodiments, the antibody, or antigen binding fragment thereof,comprises the VH region amino acid sequence set forth in SEQ ID NO: 318,321, or 360 paired with a VL region amino acid sequences selected fromthe group consisting of: SEQ ID NO: 369-453.

In certain embodiments, the antibody, or antigen binding fragmentthereof, comprises one or more CDR amino acid sequence selected from thegroup consisting of SEQ ID NO: 1-317, wherein the one or more CDR regionamino acid sequences comprises at least one or more conservative aminoacid substitutions (e.g., 1, 2, 3, 4, or 5 conservative amino acidsubstitutions). Conservative amino acid substitutions include thesubstitution of an amino acid in one class by an amino acid of the sameclass, where a class is defined by common physicochemical amino acidside chain properties and high substitution frequencies in homologousproteins found in nature, as determined, for example, by a standardDayhoff frequency exchange matrix or BLOSUM matrix. Six general classesof amino acid side chains have been categorized and include: Class I(Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln,Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and ClassVI (Phe, Tyr, Trp). For example, substitution of an Asp for anotherclass III residue such as Asn, Gln, or Glu, is a conservativesubstitution. Thus, a predicted nonessential amino acid residue in ananti-PDGFRβ antibody is preferably replaced with another amino acidresidue from the same class. Methods of identifying amino acidconservative substitutions which do not eliminate antigen binding arewell-known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187(1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burkset al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

In another embodiment, the present invention provides anti-PDGFRβantibodies, or antigen binding fragment thereof, that comprise a VHand/or VL region amino acid sequence with about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, identity tothe VH region amino acid sequence set forth in SEQ ID NO: 318-368,and/or the VL region amino acid sequence set forth in SEQ ID NO:369-453, respectively.

In another aspect, the present invention provides anti-PDGFRβ antibodiesthat bind to the same epitope and/or cross compete with an antibody, orantigen binding fragment thereof comprising the VH domain amino acidsequence set forth in SEQ ID NO: 318. Such antibodies can be identifiedusing routine competition binding assays including, for example, surfaceplasmon resonance (SPR)-based competition assays.

In another aspect, the present invention provides a diverse library ofunpaired VH domains wherein each member of the library bindsspecifically to human PDGFRβ and wherein diversity lies in the FR1-FR3regions. In a preferred embodiment, each member of the library comprisesan identical heavy chain CDR3 (e.g., the amino acid sequence set forthin SEQ ID NO: 1) amino acid sequence that binds specifically to humanPDGFRβ, and wherein diversity lies in the FR1-FR3 regions.

In another aspect, the present invention provides a diverse library ofstable VH/VL pairs wherein each member of the library binds to humanPDGFRβ Preferably each member of the library comprises a VH domaincomprising the CDR3 amino acid sequence set forth in SEQ ID NO: 1. Thestable VH/VL pairs can be selected using any methods known in the artincluding, without limitation those set forth in U.S. provisional patentapplication 61/453,106, which is hereby incorporated by reference in itsentirety.

Any type of VH or VL domain expression library can be employed in themethods of the invention. Suitable expression libraries include, withoutlimitation, nucleic acid display, phage display, and cell surfacedisplay libraries (e.g., yeast, mammalian, and bacterial cells). In apreferred embodiment, the library is a nucleic acid display librarygenerated according to the methods set forth in WO2010/011944, which ishereby incorporated by reference in its entirety. Methods for screeningexpression libraries are well known in the art. See, for example,Antibody Engineering: Methods and Protocols. Methods in MolecularBiology Volume 248, (B.K.C. Lo, Ed) Humana Press, 2004 (ISBN:1-58829-092-1), which is hereby incorporated by reference in itsentirety.

III. MODIFIED BINDING POLYPEPTIDES

In certain embodiments, binding polypeptides of the invention maycomprise one or more modifications. Modified forms of bindingpolypeptides of the invention can be made using any techniques known inthe art.

i) Reducing Immunogenicity

In certain embodiments, binding polypeptides (e.g., antibodies orantigen binding fragments thereof) of the invention are modified toreduce their immunogenicity using art-recognized techniques. Forexample, antibodies, or fragments thereof, can be chimericized,humanized, and/or deimmunized.

In one embodiment, an antibody, or antigen binding fragments thereof, ofthe invention may be chimeric. A chimeric antibody is an antibody inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies, or fragments thereof, areknown in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi etal., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods125:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397,which are incorporated herein by reference in their entireties.Techniques developed for the production of “chimeric antibodies”(Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger etal., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454(1985)) may be employed for the synthesis of said molecules. Forexample, a genetic sequence encoding a binding specificity of a mouseanti-PDGFRβ antibody molecule may be fused together with a sequence froma human antibody molecule of appropriate biological activity. As usedherein, a chimeric antibody is a molecule in which different portionsare derived from different animal species, such as those having avariable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region, e.g., humanized antibodies.

In another embodiment, an antibody, or antigen binding portion thereof,of the invention is humanized. Humanized antibodies have a bindingspecificity comprising one or more complementarity determining regions(CDRs) from a non-human antibody and framework regions from a humanantibody molecule. Often, framework residues in the human frameworkregions will be substituted with the corresponding residue from the CDRdonor antibody to alter, preferably improve, antigen binding. Theseframework substitutions are identified by methods well known in the art,e.g., by modeling of the interactions of the CDR and framework residuesto identify framework residues important for antigen binding andsequence comparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties.) Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

In some embodiments, de-immunization can be used to decrease theimmunogenicity of PDGFRβ binding polypeptides (e.g., antibody, orantigen binding portion thereof). As used herein, the term“de-immunization” includes alteration of polypeptide (e.g., an antibody,or antigen binding portion thereof) to modify T cell epitopes (see,e.g., WO9852976A1, WO0034317A2). For example, VH and VL sequences fromthe starting PDGFRβ-specific antibody, or antigen binding portionthereof, of the invention may be analyzed and a human T cell epitope“map” may be generated from each V region showing the location ofepitopes in relation to complementarity-determining regions (CDRs) andother key residues within the sequence. Individual T cell epitopes fromthe T cell epitope map are analyzed in order to identify alternativeamino acid substitutions with a low risk of altering activity of thefinal antibody. A range of alternative VH and VL sequences are designedcomprising combinations of amino acid substitutions and these sequencesare subsequently incorporated into a range of PDGFRβ-specific antibodiesor fragments thereof for use in the diagnostic and treatment methodsdisclosed herein, which are then tested for function. Typically, between12 and 24 variant antibodies are generated and tested. Complete heavyand light chain genes comprising modified V and human C regions are thencloned into expression vectors and the subsequent plasmids introducedinto cell lines for the production of whole antibody. The antibodies arethen compared in appropriate biochemical and biological assays, and theoptimal variant is identified.

ii) Effector Functions and Fc Modifications

Binding polypeptides of the invention may comprise an antibody constantregion (e.g. an IgG constant region e.g., a human IgG constant region,e.g., a human IgG1 or IgG4 constant region) which mediates one or moreeffector functions. For example, binding of the Cl component ofcomplement to an antibody constant region may activate the complementsystem. Activation of complement is important in the opsonisation andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and may also be involved in autoimmunehypersensitivity. Further, antibodies bind to receptors on various cellsvia the Fc region, with a Fc receptor binding site on the antibody Fcregion binding to a Fc receptor (FcR) on a cell. There are a number ofFc receptors which are specific for different classes of antibody,including IgG (gamma receptors), IgE (epsilon receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of antibody to Fc receptorson cell surfaces triggers a number of important and diverse biologicalresponses including engulfment and destruction of antibody-coatedparticles, clearance of immune complexes, lysis of antibody-coatedtarget cells by killer cells (called antibody-dependent cell-mediatedcytotoxicity, or ADCC), release of inflammatory mediators, placentaltransfer and control of immunoglobulin production. In preferredembodiments, the binding polypeptides (e.g., antibodies or antigenbinding fragments thereof) of the invention bind to an Fc-gammareceptor. In alternative embodiments, binding polypeptides of theinvention may comprise a constant region which is devoid of one or moreeffector functions (e.g., ADCC activity) and/or is unable to bind Fcγreceptor.

Certain embodiments of the invention include anti-PDGFRβ antibodies inwhich at least one amino acid in one or more of the constant regiondomains has been deleted or otherwise altered so as to provide desiredbiochemical characteristics such as reduced or enhanced effectorfunctions, the ability to non-covalently dimerize, increased ability tolocalize at the site of a tumor, reduced serum half-life, or increasedserum half-life when compared with a whole, unaltered antibody ofapproximately the same immunogenicity. For example, certain antibodies,or fragments thereof, for use in the diagnostic and treatment methodsdescribed herein are domain deleted antibodies which comprise apolypeptide chain similar to an immunoglobulin heavy chain, but whichlack at least a portion of one or more heavy chain domains. Forinstance, in certain antibodies, one entire domain of the constantregion of the modified antibody will be deleted, for example, all orpart of the CH2 domain will be deleted.

In certain other embodiments, binding polypeptides comprise constantregions derived from different antibody isotypes (e.g., constant regionsfrom two or more of a human IgG1, IgG2, IgG3, or IgG4). In otherembodiments, binding polypeptides comprises a chimeric hinge (i.e., ahinge comprising hinge portions derived from hinge domains of differentantibody isotypes, e.g., an upper hinge domain from an IgG4 molecule andan IgG1 middle hinge domain). In one embodiment, binding polypeptidescomprise an Fc region or portion thereof from a human IgG4 molecule anda Ser228Pro mutation (EU numbering) in the core hinge region of themolecule.

In certain embodiments, the Fc portion may be mutated to increase ordecrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain may reduce Fc receptor binding of thecirculating modified antibody thereby increasing tumor localization. Inother cases it may be that constant region modifications consistent withthe instant invention moderate complement binding and thus reduce theserum half life and nonspecific association of a conjugated cytotoxin.Yet other modifications of the constant region may be used to modifydisulfide linkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or flexibility. Theresulting physiological profile, bioavailability and other biochemicaleffects of the modifications, such as tumor localization,biodistribution and serum half-life, may easily be measured andquantified using well know immunological techniques without undueexperimentation.

In certain embodiments, an Fc domain employed in an antibody of theinvention is an Fc variant. As used herein, the term “Fc variant” refersto an Fc domain having at least one amino acid substitution relative tothe wild-type Fc domain from which said Fc domain is derived. Forexample, wherein the Fc domain is derived from a human IgG1 antibody,the Fc variant of said human IgG1 Fc domain comprises at least one aminoacid substitution relative to said Fc domain.

The amino acid substitution(s) of an Fc variant may be located at anyposition (i.e., any EU convention amino acid position) within the Fcdomain. In one embodiment, the Fc variant comprises a substitution at anamino acid position located in a hinge domain or portion thereof. Inanother embodiment, the Fc variant comprises a substitution at an aminoacid position located in a CH2 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH3 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH4 domain or portion thereof.

The binding polypeptides of the invention may employ any art-recognizedFc variant which is known to impart an improvement (e.g., reduction orenhancement) in effector function and/or FcR binding. Said Fc variantsmay include, for example, any one of the amino acid substitutionsdisclosed in International PCT Publications WO88/07089A1, WO96/14339A1,WO98/05787A1, WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2,WO00/32767A1, WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2,WO04/016750A2, WO04/029207A2, WO04/035752A2, WO04/063351A2,WO04/074455A2, WO04/099249A2, WO05/040217A2, WO05/070963A1,WO05/077981A2, WO05/092925A2, WO05/123780A2, WO06/019447A1,WO06/047350A2, and WO06/085967A2 or U.S. Pat. Nos. 5,648,260; 5,739,277;5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195;6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and7,083,784, each of which is incorporated by reference herein. In oneexemplary embodiment, a binding polypeptide of the invention maycomprise an Fc variant comprising an amino acid substitution at EUposition 268 (e.g., H268D or H268E). In another exemplary embodiment, abinding polypeptide of the invention may comprise an amino acidsubstitution at EU position 239 (e.g., S239D or S239E) and/or EUposition 332 (e.g., I332D or I332Q).

In certain embodiments, a binding polypeptide of the invention maycomprise an Fc variant comprising an amino acid substitution whichalters the antigen-independent effector functions of the antibody, inparticular the circulating half-life of the binding polypeptide. Suchbinding polypeptides exhibit either increased or decreased binding toFcRn when compared to binding polypeptides lacking these substitutions,therefore, have an increased or decreased half-life in serum,respectively. Fc variants with improved affinity for FcRn areanticipated to have longer serum half-lives, and such molecules haveuseful applications in methods of treating mammals where long half-lifeof the administered antibody is desired, e.g., to treat a chronicdisease or disorder. In contrast, Fc variants with decreased FcRnbinding affinity are expected to have shorter half-lives, and suchmolecules are also useful, for example, for administration to a mammalwhere a shortened circulation time may be advantageous, e.g. for in vivodiagnostic imaging or in situations where the starting antibody hastoxic side effects when present in the circulation for prolongedperiods. Fc variants with decreased FcRn binding affinity are also lesslikely to cross the placenta and, thus, are also useful in the treatmentof diseases or disorders in pregnant women. In addition, otherapplications in which reduced FcRn binding affinity may be desiredinclude those applications in which localization the brain, kidney,and/or liver is desired. In one exemplary embodiment, the alteredbinding polypeptides (e.g., antibodies or antigen binding fragmentsthereof) of the invention exhibit reduced transport across theepithelium of kidney glomeruli from the vasculature. In anotherembodiment, the altered binding polypeptides (e.g., antibodies orantigen binding fragments thereof) of the invention exhibit reducedtransport across the blood brain barrier (BBB) from the brain, into thevascular space. In one embodiment, an antibody with altered FcRn bindingcomprises an Fc domain having one or more amino acid substitutionswithin the “FcRn binding loop” of an Fc domain. The FcRn binding loop iscomprised of amino acid residues 280-299 (according to EU numbering).Exemplary amino acid substitutions which altered FcRn binding activityare disclosed in International PCT Publication No. WO05/047327 which isincorporated by reference herein. In certain exemplary embodiments, thebinding polypeptides (e.g., antibodies or antigen binding fragmentsthereof) of the invention comprise an Fc domain having one or more ofthe following substitutions: V284E, H285E, N286D, K290E and S304D (EUnumbering).

In other embodiments, binding polypeptides, for use in the diagnosticand treatment methods described herein have a constant region, e.g., anIgG1 or IgG4 heavy chain constant region, which is altered to reduce oreliminate glycosylation. For example, a binding polypeptides (e.g.,antibodies or antigen binding fragments thereof) of the invention mayalso comprise an Fc variant comprising an amino acid substitution whichalters the glycosylation of the antibody Fc. For example, said Fcvariant may have reduced glycosylation (e.g., N- or O-linkedglycosylation). In exemplary embodiments, the Fc variant comprisesreduced glycosylation of the N-linked glycan normally found at aminoacid position 297 (EU numbering). In another embodiment, the antibodyhas an amino acid substitution near or within a glycosylation motif, forexample, an N-linked glycosylation motif that contains the amino acidsequence NXT or NXS. In a particular embodiment, the antibody comprisesan Fc variant with an amino acid substitution at amino acid position 228or 299 (EU numbering). In more particular embodiments, the antibodycomprises an IgG1 or IgG4 constant region comprising an S228P and aT299A mutation (EU numbering).

Exemplary amino acid substitutions which confer reduce or alteredglycosylation are disclosed in International PCT Publication No.WO05/018572, which is incorporated by reference herein. In preferredembodiments, the antibodies, or fragments thereof, of the invention aremodified to eliminate glycosylation. Such antibodies, or fragmentsthereof, may be referred to as “agly” antibodies, or fragments thereof,(e.g. “agly” antibodies). While not being bound by theory, it isbelieved that “agly” antibodies, or fragments thereof, may have animproved safety and stability profile in vivo. Exemplary aglyantibodies, or fragments thereof, comprise an aglycosylated Fc region ofan IgG4 antibody which is devoid of Fc-effector function therebyeliminating the potential for Fc mediated toxicity to the normal vitalorgans that express PDGFRβ. In yet other embodiments, antibodies, orfragments thereof, of the invention comprise an altered glycan. Forexample, the antibody may have a reduced number of fucose residues on anN-glycan at Asn297 of the Fc region, i.e., is afucosylated. In anotherembodiment, the antibody may have an altered number of sialic acidresidues on the N-glycan at Asn297 of the Fc region.

iii) Covalent Attachment

Binding polypeptides of the invention may be modified, e.g., by thecovalent attachment of a molecule to the binding polypeptide such thatcovalent attachment does not prevent the binding polypeptide fromspecifically binding to its cognate epitope. For example, but not by wayof limitation, the antibodies, or fragments thereof, of the inventionmay be modified by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, etc. Additionally, the derivativemay contain one or more non-classical amino acids.

Binding polypeptide (e.g., antibodies, or fragments thereof) of theinvention may further be recombinantly fused to a heterologouspolypeptide at the N- or C-terminus or chemically conjugated (includingcovalent and non-covalent conjugations) to polypeptides or othercompositions. For example, anti-PDGFRβ antibodies may be recombinantlyfused or conjugated to molecules useful as labels in detection assaysand effector molecules such as heterologous polypeptides, drugs,radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.

Binding polypeptides may be fused to heterologous polypeptides toincrease the in vivo half life or for use in immunoassays using methodsknown in the art. For example, in one embodiment, PEG can be conjugatedto the binding polypeptides of the invention to increase their half-lifein vivo. Leong, S. R., et al., Cytokine 16:106 (2001); Adv. in DrugDeliv. Rev. 54:531 (2002); or Weir et al., Biochem. Soc. Transactions30:512 (2002).

Moreover, binding polypeptides of the invention can be fused to markersequences, such as a peptide to facilitate their purification ordetection. In preferred embodiments, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

Binding polypeptides of the invention may be used in non-conjugated formor may be conjugated to at least one of a variety of molecules, e.g., toimprove the therapeutic properties of the molecule, to facilitate targetdetection, or for imaging or therapy of the patient. Bindingpolypeptides of the invention can be labeled or conjugated either beforeor after purification, when purification is performed. In particular,binding polypeptides of the invention may be conjugated to therapeuticagents, prodrugs, peptides, proteins, enzymes, viruses, lipids,biological response modifiers, pharmaceutical agents, or PEG.

The present invention further encompasses binding polypeptides of theinvention conjugated to a diagnostic or therapeutic agent. The bindingpolypeptides can be used diagnostically to, for example, monitor thedevelopment or progression of a immune cell disorder (e.g., CLL) as partof a clinical testing procedure to, e.g., determine the efficacy of agiven treatment and/or prevention regimen. Detection can be facilitatedby coupling the binding polypeptides to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. See,for example, U.S. Pat. No. 4,741,900 for metal ions which can beconjugated to antibodies for use as diagnostics according to the presentinvention. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, .beta.-galactosidase, or acetylcholinesterase;examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I, 111In or 99Tc.

Binding polypeptides for use in the diagnostic and treatment methodsdisclosed herein may be conjugated to cytotoxins (such as radioisotopes,cytotoxic drugs, or toxins) therapeutic agents, cytostatic agents,biological toxins, prodrugs, peptides, proteins, enzymes, viruses,lipids, biological response modifiers, pharmaceutical agents,immunologically active ligands (e.g., lymphokines or other antibodieswherein the resulting molecule binds to both the neoplastic cell and aneffector cell such as a T cell), or PEG.

In another embodiment, an anti-PDGFRβ antibody for use in the diagnosticand treatment methods disclosed herein can be conjugated to a moleculethat decreases tumor cell growth. In other embodiments, the disclosedcompositions may comprise antibodies, or fragments thereof, coupled todrugs or prodrugs. Still other embodiments of the present inventioncomprise the use of antibodies, or fragments thereof, conjugated tospecific biotoxins or their cytotoxic fragments such as ricin, gelonin,Pseudomonas exotoxin or diphtheria toxin. The selection of whichconjugated or unconjugated antibodyto use will depend on the type andstage of cancer, use of adjunct treatment (e.g., chemotherapy orexternal radiation) and patient condition. It will be appreciated thatone skilled in the art could readily make such a selection in view ofthe teachings herein.

It will be appreciated that, in previous studies, anti-tumor antibodieslabeled with isotopes have been used successfully to destroy tumor cellsin animal models, and in some cases in humans. Exemplary radioisotopesinclude: 90Y, 125I, 131I, 123I, 111In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho,177Lu, 186Re and 188Re. The radionuclides act by producing ionizingradiation which causes multiple strand breaks in nuclear DNA, leading tocell death. The isotopes used to produce therapeutic conjugatestypically produce high energy alpha- or beta-particles which have ashort path length. Such radionuclides kill cells to which they are inclose proximity, for example neoplastic cells to which the conjugate hasattached or has entered. They have little or no effect on non-localizedcells. Radionuclides are essentially non-immunogenic.

IV. EXPRESSION OF BINDING POLYPEPTIDES

Following manipulation of the isolated genetic material to providebinding polypeptides of the invention as set forth above, the genes aretypically inserted in an expression vector for introduction into hostcells that may be used to produce the desired quantity of the claimedantibodies, or fragments thereof.

The term “vector” or “expression vector” is used herein for the purposesof the specification and claims, to mean vectors used in accordance withthe present invention as a vehicle for introducing into and expressing adesired gene in a cell. As known to those skilled in the art, suchvectors may easily be selected from the group consisting of plasmids,phages, viruses and retroviruses. In general, vectors compatible withthe instant invention will comprise a selection marker, appropriaterestriction sites to facilitate cloning of the desired gene and theability to enter and/or replicate in eukaryotic or prokaryotic cells.

Numerous expression vector systems may be employed for the purposes ofthis invention. For example, one class of vector utilizes DNA elementswhich are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV) or SV40 virus. Others involve the use ofpolycistronic systems with internal ribosome binding sites.Additionally, cells which have integrated the DNA into their chromosomesmay be selected by introducing one or more markers which allow selectionof transfected host cells. The marker may provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation. Additional elements may also beneeded for optimal synthesis of mRNA. These elements may include signalsequences, splice signals, as well as transcriptional promoters,enhancers, and termination signals. In particularly preferredembodiments the cloned variable region genes are inserted into anexpression vector along with the heavy and light chain constant regiongenes (preferably human) synthetic as discussed above.

In other preferred embodiments the binding polypeptides, or fragmentsthereof, of the invention may be expressed using polycistronicconstructs. In such expression systems, multiple gene products ofinterest such as heavy and light chains of antibodies may be producedfrom a single polycistronic construct. These systems advantageously usean internal ribosome entry site (IRES) to provide relatively high levelsof polypeptides of the invention in eukaryotic host cells. CompatibleIRES sequences are disclosed in U.S. Pat. No. 6,193,980 which isincorporated herein. Those skilled in the art will appreciate that suchexpression systems may be used to effectively produce the full range ofpolypeptides disclosed in the instant application.

More generally, once a vector or DNA sequence encoding an antibody, orfragment thereof, has been prepared, the expression vector may beintroduced into an appropriate host cell. That is, the host cells may betransformed. Introduction of the plasmid into the host cell can beaccomplished by various techniques well known to those of skill in theart. These include, but are not limited to, transfection (includingelectrophoresis and electroporation), protoplast fusion, calciumphosphate precipitation, cell fusion with enveloped DNA, microinjection,and infection with intact virus. See, Ridgway, A. A. G. “MammalianExpression Vectors” Chapter 24.2, pp. 470-472 Vectors, Rodriguez andDenhardt, Eds. (Butterworths, Boston, Mass. 1988). Most preferably,plasmid introduction into the host is via electroporation. Thetransformed cells are grown under conditions appropriate to theproduction of the light chains and heavy chains, and assayed for heavyand/or light chain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orflourescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

As used herein, the term “transformation” shall be used in a broad senseto refer to the introduction of DNA into a recipient host cell thatchanges the genotype and consequently results in a change in therecipient cell.

Along those same lines, “host cells” refers to cells that have beentransformed with vectors constructed using recombinant DNA techniquesand encoding at least one heterologous gene. In descriptions ofprocesses for isolation of polypeptides from recombinant hosts, theterms “cell” and “cell culture” are used interchangeably to denote thesource of antibody unless it is clearly specified otherwise. In otherwords, recovery of polypeptide from the “cells” may mean either fromspun down whole cells, or from the cell culture containing both themedium and the suspended cells.

In one embodiment, the host cell line used for antibody expression is ofmammalian origin; those skilled in the art can determine particular hostcell lines which are best suited for the desired gene product to beexpressed therein. Exemplary host cell lines include, but are notlimited to, DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus),HELA (human cervical carcinoma), CVI (monkey kidney line), COS (aderivative of CVI with SV40 T antigen), R1610 (Chinese hamsterfibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line),SP2/0 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI(human lymphocyte), 293 (human kidney). In one embodiment, the cell lineprovides for altered glycosylation, e.g., afucosylation, of theantibodyexpressed therefrom (e.g., PER.C6® (Crucell) or FUT8-knock-outCHO cell lines (Potelligent® Cells) (Biowa, Princeton, N.J.)). In oneembodiment NSO cells may be used. CHO cells are particularly preferred.Host cell lines are typically available from commercial services, theAmerican Tissue Culture Collection or from published literature.

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose and/or (immuno-)affinity chromatography.

Genes encoding the binding polypeptides, or fragments thereof, of theinvention can also be expressed non-mammalian cells such as bacteria oryeast or plant cells. In this regard it will be appreciated that variousunicellular non-mammalian microorganisms such as bacteria can also betransformed; i.e. those capable of being grown in cultures orfermentation. Bacteria, which are susceptible to transformation, includemembers of the enterobacteriaceae, such as strains of Escherichia colior Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus;Streptococcus, and Haemophilus influenzae. It will further beappreciated that, when expressed in bacteria, the polypeptides canbecome part of inclusion bodies. The polypeptides must be isolated,purified and then assembled into functional molecules.

In addition to prokaryotes, eukaryotic microbes may also be used.

Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available. For expression in Saccharomyces, the plasmidYRp7, for example, (Stinchcomb et al., Nature, 282:39 (1979); Kingsmanet al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) iscommonly used. This plasmid already contains the TRP1 gene whichprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1(Jones, Genetics, 85:12 (1977)). The presence of the trpl lesion as acharacteristic of the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan.

V. PHARMACEUTICAL FORMULATIONS AND METHODS OF ADMINISTRATION OF BINDINGPOLYPEPTIDES

In another aspect, the invention provides pharmaceutical compositionscomprising an anti-PDGFRβ antibody, or fragment thereof.

Methods of preparing and administering antibodies, or fragments thereof,of the invention to a subject are well known to or are readilydetermined by those skilled in the art. The route of administration ofthe antibodies, or fragments thereof, of the invention may be oral,parenteral, by inhalation or topical. The term parenteral as used hereinincludes intravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, rectal or vaginal administration. The intravenous,intraarterial, subcutaneous and intramuscular forms of parenteraladministration are generally preferred. While all these forms ofadministration are clearly contemplated as being within the scope of theinvention, a form for administration would be a solution for injection,in particular for intravenous or intraarterial injection or drip.Usually, a suitable pharmaceutical composition for injection maycomprise a buffer (e.g. acetate, phosphate or citrate buffer), asurfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. humanalbumin), etc. However, in other methods compatible with the teachingsherein, the polypeptides can be delivered directly to the site of theadverse cellular population thereby increasing the exposure of thediseased tissue to the therapeutic agent.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the subject invention, pharmaceutically acceptable carriersinclude, but are not limited to, 0.01-0.1M and preferably 0.05Mphosphate buffer or 0.8% saline. Other common parenteral vehiclesinclude sodium phosphate solutions, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's, or fixed oils. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives may also be present such as for example, antimicrobials,antioxidants, chelating agents, and inert gases and the like. Moreparticularly, pharmaceutical compositions suitable for injectable useinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersions. In such cases, the composition mustbe sterile and should be fluid to the extent that easy syringabilityexists. It should be stable under the conditions of manufacture andstorage and will preferably be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal and the like. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols, such as mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

In any case, sterile injectable solutions can be prepared byincorporating an active compound (e.g., an antibody by itself or incombination with other active agents) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedherein, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle, which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying,which yields a powder of an active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The preparations for injections are processed, filled into containerssuch as ampoules, bags, bottles, syringes or vials, and sealed underaseptic conditions according to methods known in the art. Further, thepreparations may be packaged and sold in the form of a kit such as thosedescribed in co-pending U.S. Ser. No. 09/259,337 and U.S. Ser. No.09/259,338 each of which is incorporated herein by reference. Sucharticles of manufacture will preferably have labels or package insertsindicating that the associated compositions are useful for treating asubject suffering from, or predisposed to autoimmune or neoplasticdisorders.

Effective doses of the stabilized antibodies, or fragments thereof, ofthe present invention, for the treatment of the above describedconditions vary depending upon many different factors, including meansof administration, target site, physiological state of the patient,whether the patient is human or an animal, other medicationsadministered, and whether treatment is prophylactic or therapeutic.Usually, the patient is a human, but non-human mammals includingtransgenic mammals can also be treated. Treatment dosages may betitrated using routine methods known to those of skill in the art tooptimize safety and efficacy.

For passive immunization with an antibody of the invention, the dosagemay range, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1mg/kg, 2 mg/kg, etc.), of the host body weight. For example dosages canbe 1 mg/kg body weight or 10 mg/kg body weight or within the range of1-10 mg/kg, preferably at least 1 mg/kg. Doses intermediate in the aboveranges are also intended to be within the scope of the invention.

Subjects can be administered such doses daily, on alternative days,weekly or according to any other schedule determined by empiricalanalysis. An exemplary treatment entails administration in multipledosages over a prolonged period, for example, of at least six months.Additional exemplary treatment regimes entail administration once perevery two weeks or once a month or once every 3 to 6 months. Exemplarydosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or moremonoclonal antibodies with different binding specificities areadministered simultaneously, in which case the dosage of each antibodyadministered may fall within the ranges indicated.

Antibodies, or fragments thereof, of the invention can be administeredon multiple occasions. Intervals between single dosages can be, e.g.,daily, weekly, monthly or yearly. Intervals can also be irregular asindicated by measuring blood levels of polypeptide or target molecule inthe patient. In some methods, dosage is adjusted to achieve a certainplasma antibody or toxin concentration, e.g., 1-1000 ug/ml or 25-300ug/ml. Alternatively, antibodies, or fragments thereof, can beadministered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the antibody in the patient. In general, humanizedantibodies show the longest half-life, followed by chimeric antibodiesand nonhuman antibodies. In one embodiment, the antibodies, or fragmentsthereof, of the invention can be administered in unconjugated form. Inanother embodiment, the antibodies of the invention can be administeredmultiple times in conjugated form. In still another embodiment, theantibodies, or fragments thereof, of the invention can be administeredin unconjugated form, then in conjugated form, or vise versa.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, compositions containing the present antibodies or acocktail thereof are administered to a patient not already in thedisease state to enhance the patient's resistance. Such an amount isdefined to be a “prophylactic effective dose.” In this use, the preciseamounts again depend upon the patient's state of health and generalimmunity, but generally range from 0.1 to 25 mg per dose, especially 0.5to 2.5 mg per dose. A relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives.

In therapeutic applications, a relatively high dosage (e.g., from about1 to 400 mg/kg of antibody per dose, with dosages of from 5 to 25 mgbeing more commonly used for radioimmunoconjugates and higher doses forcytotoxin-drug conjugated molecules) at relatively short intervals issometimes required until progression of the disease is reduced orterminated, and preferably until the patient shows partial or completeamelioration of symptoms of disease. Thereafter, the patent can beadministered a prophylactic regime.

In one embodiment, a subject can be treated with a nucleic acid moleculeencoding a polypeptide of the invention (e.g., in a vector). Doses fornucleic acids encoding polypeptides range from about 10 ng to 1 g, 100ng to 100 mg, 1 ug to 10 mg, or 30-300 ug DNA per patient. Doses forinfectious viral vectors vary from 10-100, or more, virions per dose.

Therapeutic agents can be administered by parenteral, topical,intravenous, oral, subcutaneous, intraarterial, intracranial,intraperitoneal, intranasal or intramuscular means for prophylacticand/or therapeutic treatment. Intramuscular injection or intravenousinfusion are preferred for administration of a antibody of theinvention. In some methods, therapeutic antibodies, or fragmentsthereof, are injected directly into the cranium. In some methods,antibodies, or fragments thereof, are administered as a sustainedrelease composition or device, such as a Medipad™ device.

Agents of the invention can optionally be administered in combinationwith other agents that are effective in treating the disorder orcondition in need of treatment (e.g., prophylactic or therapeutic).Preferred additional agents are those which are art recognized and arestandardly administered for a particular disorder.

Effective single treatment dosages (i.e., therapeutically effectiveamounts) of 90Y-labeled antibodies of the invention range from betweenabout 5 and about 75 mCi, more preferably between about 10 and about 40mCi. Effective single treatment non-marrow ablative dosages of131I-labeled antibodies range from between about 5 and about 70 mCi,more preferably between about 5 and about 40 mCi. Effective singletreatment ablative dosages (i.e., may require autologous bone marrowtransplantation) of 131I-labeled antibodies range from between about 30and about 600 mCi, more preferably between about 50 and less than about500 mCi. In conjunction with a chimeric modified antibody, owing to thelonger circulating half life vis-a-vis murine antibodies, an effectivesingle treatment non-marrow ablative dosages of iodine-131 labeledchimeric antibodies range from between about 5 and about 40 mCi, morepreferably less than about 30 mCi. Imaging criteria for, e.g., the 111Inlabel, are typically less than about 5 mCi.

While a great deal of clinical experience has been gained with 131I and0.90Y, other radiolabels are known in the art and have been used forsimilar purposes. Still other radioisotopes are used for imaging. Forexample, additional radioisotopes which are compatible with the scope ofthe instant invention include, but are not limited to, 123I, 125I, 32P,57Co, 64Cu, 67Cu, 77Br, 81Rb, 81Kr, 87Sr, 113In, 127Cs, 129Cs, 132I,197Hg, 203Pb, 206Bi, 177Lu, 186Re, 212Pb, 212Bi, 47Sc, 105Rh, 109Pd,153Sm, 188Re, 199Au, 225Ac, 211A 213Bi. In this respect alpha, gamma andbeta emitters are all compatible with in the instant invention. Further,in view of the instant disclosure it is submitted that one skilled inthe art could readily determine which radionuclides are compatible witha selected course of treatment without undue experimentation. To thisend, additional radionuclides which have already been used in clinicaldiagnosis include 125I, 123I, 99Tc, 43K, 52Fe, 67Ga, 68Ga, as well as111In. Antibodies have also been labeled with a variety of radionuclidesfor potential use in targeted immunotherapy (Peirersz et al. Immunol.Cell Biol. 65: 111-125 (1987)). These radionuclides include 188Re and186Re as well as 199Au and 67Cu to a lesser extent. U.S. Pat. No.5,460,785 provides additional data regarding such radioisotopes and isincorporated herein by reference.

As previously discussed, the antibodies, or fragments thereof, of theinvention, can be administered in a pharmaceutically effective amountfor the in vivo treatment of mammalian disorders. In this regard, itwill be appreciated that the disclosed antibodies, or fragments thereof,will be formulated so as to facilitate administration and promotestability of the active agent. Preferably, pharmaceutical compositionsin accordance with the present invention comprise a pharmaceuticallyacceptable, non-toxic, sterile carrier such as physiological saline,non-toxic buffers, preservatives and the like. For the purposes of theinstant application, a pharmaceutically effective amount of a antibodyof the invention, conjugated or unconjugated to a therapeutic agent,shall be held to mean an amount sufficient to achieve effective bindingto a target and to achieve a benefit, e.g., to ameliorate symptoms of adisease or disorder or to detect a substance or a cell. In the case oftumor cells, the polypeptide will be preferably be capable ofinteracting with selected immunoreactive antigens on neoplastic orimmunoreactive cells and provide for an increase in the death of thosecells. Of course, the pharmaceutical compositions of the presentinvention may be administered in single or multiple doses to provide fora pharmaceutically effective amount of the polypeptide.

In keeping with the scope of the present disclosure, the antibodies ofthe invention may be administered to a human or other animal inaccordance with the aforementioned methods of treatment in an amountsufficient to produce a therapeutic or prophylactic effect. Thepolypeptides of the invention can be administered to such human or otheranimal in a conventional dosage form prepared by combining the antibodyof the invention with a conventional pharmaceutically acceptable carrieror diluent according to known techniques. It will be recognized by oneof skill in the art that the form and character of the pharmaceuticallyacceptable carrier or diluent is dictated by the amount of activeingredient with which it is to be combined, the route of administrationand other well-known variables. Those skilled in the art will furtherappreciate that a cocktail comprising one or more species ofpolypeptides according to the present invention may prove to beparticularly effective.

VI. METHODS OF TREATING PDGFRβ-ASSOCIATED DISEASE OR DISORDERS

The binding polypeptides, or fragments thereof, of the invention areuseful for antagonizing PDGFRβ activity. Accordingly, in another aspect,the invention provides methods for treating PDGFRβ-associated diseasesor disorders by administering to a subject in need of thereof apharmaceutical composition comprising one or more anti-PDGFRβ antibody,or antigen binding fragment thereof of the invention.

PDGFRβ-associated diseases or disorders amenable to treatment include,without limitation: Age related macular degeneration (AMD); restenosis,including coronary restenosis after angioplasty, atherectomy, or otherinvasive methods of plaque removal, and renal or peripheral arteryrestenosis after the same procedures; vascular proliferative phenomenaand fibrosis associated with other forms of acute injury such as:pulmonary fibrosis associated with adult respiratory distress syndrome,renal fibrosis associated with nephritis, coronary stenosis associatedwith Kawasake's disease, and vascular narrowings associated with otherarteritides such as Takayasha's disease; fibrotic processes, such asscleroderma, myofibrosis; and cancer (e.g., tumor cell proliferation andneovascularization)

One skilled in the art would be able, by routine experimentation, todetermine what an effective, non-toxic amount of antibody (or additionaltherapeutic agent) would be for the purpose of treating aPDGFRβ-associated disease or disorder. For example, a therapeuticallyactive amount of a polypeptide may vary according to factors such as thedisease stage (e.g., stage I versus stage IV), age, sex, medicalcomplications (e.g., immunosuppressed conditions or diseases) and weightof the subject, and the ability of the antibody to elicit a desiredresponse in the subject. The dosage regimen may be adjusted to providethe optimum therapeutic response. For example, several divided doses maybe administered daily, or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. Generally,however, an effective dosage is expected to be in the range of about0.05 to 100 milligrams per kilogram body weight per day and morepreferably from about 0.5 to 10, milligrams per kilogram body weight perday.

VII. EXAMPLES

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents ofSequence Listing, figures and all references, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

Example 1. Isolation of VH Domains that Bind Specifically to HumanPDGFRβ

VH domains that bind specifically to human PDGFRβ were selected usingDNA display as set forth in WO2010/011944, which is hereby incorporatedby reference in its entirety. Specifically, a naïve, human VH domain DNAdisplay library derived from ten bone marrow donors was subject to sixrounds of selection against human PDGFRβ. The selected binders werecloned and sequenced. From this screen, VH domain clones A4, B4 and G2were selected, the amino acid sequences of which are set forth in Table3.

Example 2. HCDR3 Shuffling A. VH Library Construction

To screen for VH domains with improved binding characteristics, theHCDR3 sequence of clone A4 (designated XB1511) was shuffled into a naïvehuman VH library, which was further selected for binding to human andmouse PDGFRβ. Specifically, the DNA sequence coding for the HCDR3 ofclone A4 (SEQ ID NO: 1) was synthesized and assembled into a librarycomprising framework regions 1-3 of naïve human VH domains amplifiedfrom bone marrow B cells and PBMCs using framework specificoligonucleotides. Human VH framework regions 1-3 were amplified using 5′VH family-specific and 3′ generic FR3 reverse primers to create separatelibraries of VH family framework regions. The VH family frameworklibraries and the XB1511 HCDR3 were shuffled by further PCRamplification using 5′ T7TMV and 3′ XB1511 FR3CDR3FR4 oligos. This alsoadded a T7TMV promoter sequence at the 5′ end for in vitrotranscription/translation. A C-terminal C_(μ)3 sequence and a FLAG tag(for purification after translation) were also added by PCR using FR4Cu3 Reverse and Y109 primers, respectively, together with the 5′ T7TMVprimer. The nucleic acid sequences of the oligonucleotides used forpreparation of the HCDR3-shuffled VH library are set forth in Table 5. Aschematic representation of the VH library construction is set forth inFIG. 1.

TABLE 5 Oligonucleotides for constructing HCDR3 shuffled VH librariesSEQ ID Oligo Sequence NO. FR3  CGCACAGTAATACACGGC 369 Reverse VH1aCAATTACTATTTACAATTACAATGCAGGTKCAGCTGGTGCAGTCTG 370 VH1bCAATTACTATTTACAATTACAATGCAGGTCCAGCTTGTGCAGTCTG 371 VH1cCAATTACTATTTACAATTACAATGSAGGTCCAGCTGGTACAGTCTG 372 VH1dCAATTACTATTTACAATTACAATGCARATGCAGCTGGTGCAGTCTG 373 VH2CAATTACTATTTACAATTACAATGCAGRTCACCTTGAAGGAGTCTG 374 VH3aCAATTACTATTTACAATTACAATGGARGTGCAGCTGGTGGAGTCTG 375 VH3bCAATTACTATTTACAATTACAATGCAGGTGCAGCTGGTGGAGTCTG 376 VH3cCAATTACTATTTACAATTACAATGGAGGTGCAGCTGTTGGAGTCTG 377 VH4aCAATTACTATTTACAATTACAATGCAGSTGCAGCTGCAGGAG 378 VH4bCAATTACTATTTACAATTACAATGCAGGTGCAGCTACAGCAGTGG 379 VH5CAATTACTATTTACAATTACAATGGARGTGCAGCTGGTGCAGTCTG 380 VH6CAATTACTATTTACAATTACAATGCAGGTACAGCTGCAGCAGTCAG 381 VH7CAATTACTATTTACAATTACAATGCAGGTGCAGCTGGTGCAATCTG 382 T7TMVUTRTAATACGACTCACTATAGGGACAATTACTATTTACAATTACA 383 XB1511TGAGGAGACGGTGACCAGGGTTCCCTGGCCCCAGTAGCTCCTGTCG 384 FR3CDR3FR4CCCCCATGTKTCGCACAGTAATACACGGC Reverse FR4 Cu3GGAGACGAGGGGGAAAAGGGTTGAGGAGACGGTGACCAG 385 Reverse Y109TTTTTTTTTTTTTTTTTTTTAAATAGCGGATGCTAAGGACGACTTG 386TCGTCGTCGTCCTTGTAGTCGGAGACGAGGGGGAAAAGGGT

B. Library Screening

The HCDR3 shuffled VH domain library was then transcribed into an mRNAlibrary and subjected to selection with dsDNA display technology as setforth in WO2010/011944. The selection was carried out with human andmouse PDGFRβ at alternate round for 4 rounds. Kinetic controlled on- andoff-rate selection was applied at successive rounds to increase thestringency of selection, and thus select for VH domains with highaffinity for PDGFRβ. Specifically, selection was performed as follows:Round 1 (R1) with 10 nM of immobilized human PDGFRβ; R2 with immobilized100 nM mouse PDGFRβ; R3 with 10 nM soluble human PDGFRβ and competedwith 200 nM immobilized human PDGFRβ for 24 hours and 120 hours; and R4with 10 nM mouse PDGFRβ. The R4 binding pool was subcloned for DNAsequencing. Analysis of the sequences of the R4 binding pool showed thatthe HCDR3 of XB1511 was present in a variety of different frameworkcontexts. No wild type parental sequence was obtained from the set ofsequences analyzed. The amino acid sequences of the selected VH domainsare set forth in Table 3, herein.

C. Binding Specificity of Selected HCDR3 Shuffled VH Domains

The R4 binding pool selected above was assessed for binding to bothhuman and mouse PDGFRβ using a ³⁵S Met-labelled in vitro translatedlibrary. Specifically, binding of the pool to epoxy beads, 100 nM ofhuman IgG, human PDGFRβ and mouse PDGFRβ were assessed. As shown in FIG.2, the parental XB1511 VH domain showed specific binding to humanPDGFRβ, and undetectable binding to mouse PDGFRβ. The framework shuffledpre-selected library showed weak binding to human PDGFRβ. However, incontrast, the R4 framework shuffled library showed significant bindingto both human and mouse PDGFRβ.

Example 3. Identification of Stable VL/VH Pairs

A. Construction of VL DNA libraries

Human VL libraries (Vkappa and Vlamda) were constructed from B cells ofyoung healthy donors (Allcells) by RT-PCR. To ensure the diversity ofthe library, 300 million bone marrow mononuclear cells and 100 millionperipheral blood mononuclear cells were obtained from ten donors andused for naive VH and VL library construction. A schematic of thelibrary generation method is set forth in FIG. 3.

Oligonucleotide primers for cDNA synthesis and subsequent PCRamplification of the Vkappa and Vlamda sequences were designed as setforth in Table 4. Specifically, multiple sense primers were designedfrom the Vκ and Vλ FR1 regions of each family with an upstream UTRsequence. The anti-sense primers for κ and λ gene amplification weredesigned from the constant regions nested to Cκ1 (Cκ2) or Jλ with thesame Cκ2 downstream (JλCκ2). The Vκ and Vλ libraries carry the sameC-terminal sequence for PCR amplification during the selection cycles.

mRNA was prepared from individual donors using a FastTrack mRNApreparation kit (Invitrogen) following the protocol provided by the kit.First strand cDNA was synthesized from the isolated mRNA using primersspecific for the light chain kappa and lambda constant regions (Cκ1 andCλ1).

PCR amplification of the Vkappa and Vlamda sequences was performed withCκ2 and Vκ family specific or JλCκ2 mix and Vλ family specific primersusing cDNA as a template. The PCR was performed for individual Vκ and Vλfamilies and individual donors for 18-20 cycles. After gel purification,Vκ and Vλ libraries from each different source were pooled to generatethe final Vκ and Vλ libraries.

TABLE 6Oligonucleotides for constructing human Vλ and Vκ DNA display librariesOligo Sequence SEQ ID NO. Ck1 CAACTGCTCATCAGATGGCGG 387 Cl1CAGTGTGGCCTTGTTGGCTTG 388 Ck2 AGATGGTGCAGCCACAGTTCG 389 J11-3Ck2AGATGGTGCAGCCACAGTTCGTAGACGGTSASCTTGGTCCC 390 J17Ck2AGATGGTGCAGCCACAGTTCGGAGACGGTCAGCTGGGTGCC 391 T7TMVUTRTAATACGACTCACTATAGGGACAATTACTATTTACAATTACA 392 Vλ oligos UTRVk1aCAATTACTATTTACAATTACAATGRACATCCAGATGACCCAG 393 UTRVk1bCAATTACTATTTACAATTACAATGGMCATCCAGTTGACCCAG 394 UTRVk1cCAATTACTATTTACAATTACAATGGCCATCCRGATGACCCAG 395 UTRVk1dCAATTACTATTTACAATTACAATGGTCATCTGGATGACCCAG 396 UTRVk2aCAATTACTATTTACAATTACAATGGATATTGTGATGACCCAG 397 UTRVk2bCAATTACTATTTACAATTACAATGGATRTTGTGATGACTCAG 398 UTRVk3aCAATTACTATTTACAATTACAATGGAAATTGTGTTGACRCAG 399 UTRVk3bCAATTACTATTTACAATTACAATGGAAATAGTGATGACGCAG 400 UTRVk3cCAATTACTATTTACAATTACAATGGAAATTGTAATGACACAG 401 UTRVk4aCAATTACTATTTACAATTACAATGGACATCGTGATGACCCAG 402 UTRVk5aCAATTACTATTTACAATTACAATGGAAACGACACTCACGCAG 403 UTRVk6aCAATTACTATTTACAATTACAATGGAAATTGTGCTGACTCAG 404 UTRVk6bCAATTACTATTTACAATTACAATGGATGTTGTGATGACACAG 405 Vλ oligos UTRVL1aCAATTACTATTTACAATTACAATGCAGTCTGTGCTGACKCAG 406 UTRVL1bCAATTACTATTTACAATTACAATGCAGTCTGTGYTGACGCAG 407 UTRVL2CAATTACTATTTACAATTACAATGCAGTCTGCCCTGACTCAG 408 UTRVL3aCAATTACTATTTACAATTACAATGTCCTATGWGCTGACTCAG 409 UTRVL3bCAATTACTATTTACAATTACAATGTCCTATGAGCTGACACAG 410 UTRVL3cCAATTACTATTTACAATTACAATGTCTTCTGAGCTGACTCAG 411 UTRVL3dCAATTACTATTTACAATTACAATGTCCTATGAGCTGATGCAG 412 UTRVL4CAATTACTATTTACAATTACAATGCAGCYTGTGCTGACTCAA 413 UTRVL5CAATTACTATTTACAATTACAATGCAGSCTGTGCTGACTCAG 414 UTRVL6CAATTACTATTTACAATTACAATGAATTTTATGCTGACTCAG 415 UTRVL7CAATTACTATTTACAATTACAATGCAGRCTGTGGTGACTCAG 416 UTRVL8CAATTACTATTTACAATTACAATGCAGACTGTGGTGACCCAG 417 UTRVL4/9CAATTACTATTTACAATTACAATGCWGCCTGTGCTGACTCAG 418 UTRVL10CAATTACTATTTACAATTACAATGCAGGCAGGGCTGACTCAG 419R = A/G, Y = C/T, K = G/T, M = A/C, S = G/C, W = A/TB. Generation of VL Fusion Libraries by dsDNA Display

Vκ and Vλ DNA libraries generated using the methods set forth in thisExample were transcribed into mRNA libraries using the T7 Megascript kit(Invitrogen, Cat# AM1334). The mRNA was purified with RNeasy MinEluteCleanup Kit (Qiagen, Cat#74204) following protocol provided by the kit.A total of 600 pmol of RNA (300 pmol of Vκ and Vλ libraries) was ligatedand assembled with dsDNA display linkers and components as described inWO2010/011944. The assembled VL library was subjected to in vitrotranslation to create a fusion library in which each VL domain(phenotype) is stably fused to its coding sequence (genotype). ³⁵S Metwas incorporated in the translation process to radiolabel the fusions.The library was then purified with oligo dT cellulose, converted into adsDNA display library using the standard molecular biology techniques ofreverse transcription, RNaseH digestion, 2^(nd) strand DNA synthesis,followed by flag tag purification.

C. Identification of VL Pairs for XB1511, and XB2202 VH Domains

XB1511 VH domain was translated as free protein (with incorporation of³⁵S Met in the translation reaction) and affinity purified through ac-terminal flag tag. The XB1511 VH domain and a purified VL domainfusion library (prepared as above) were then mixed at an equal molarratio and incubated at 25C overnight to allow for in vitro associationof VH and VL fusion domains through their hydrophobic patch. The mixturewas then contacted with PDGFRβ target pre-immobilized on Epoxy450 beadsor in solution and captured by protein A beads, Complexes that bound tothe immobilized PDGFRβ target were washed and eluted with 0.1N KOH. PCRwas performed with VL specific primer sets to recover the VLs that boundto the PDGFRβ target, both as VH-VL pairs and as unpaired VL domains.The VL pairing was performed for 3 rounds, with low stringency (100 nMPDGFRβ) for the first 2 rounds and higher stringency (10 nM PDGFRβ) forthe third round. The XB2202 VH domain was also paired with the VLlibrary similarly for two rounds. For each round of XB2202/VL pairingand selection, the stringency was increased by kinetic controlled on andoff rate strategy to identify VL domains that paired stably with XB2202VH domain and enhance the VH binding.

VL domain pools identified above were then cloned into Blunt Zero TOPOvector (Invitrogen) and VL-encoding DNA sequences were amplified fromthe resultant bacterial colonies by PCR using M13 forward and reverseprimers. The individual amplified VL-encoding DNA sequences were thensequenced. The sequence data obtained from VL pools showed that adiverse repertoire of VLs was enriched through the process. Multiplefamilies and frameworks were present in the pool. Several VLs werepresent as replicates or families. Distinct VL families could beidentified and several VLs were present more than once. Exemplary VLsequences identified using the methods of the invention that pair withthe PDGFRβ-binding VH domains XB1511 and XB2202 are set forth in Table 4herein.

D. Evaluation of Identified VH and VL Pairs

To evaluate the characteristics of the identified VH-VL pairs, 10-12scFVs from each pool were constructed and produced by either in vitrotranslation or by E. coli expression, followed by affinity purification.

A PDGFRβ binding ELISA assay was performed to assess the binding of thescFv to immobilized PDGFRβ and to determine the EC50. Specifically, 2ug/mL of human PDGFRβ and human Fc or IgG in PBS was immobilized onMaxisorp plates at 4° C. overnight. The plate was then washed andblocked with superblock. In vitro translated crude scFv lysate wasdiluted 1:3 in 1× PBST. 100 ul of the diluted scFv lysate was loadedinto each well of Maxisorp plates and incubated for 1 hour at roomtemperature. scFv that bound to immobilized PDGFRβ was detected byanti-flag antibody-HRP at 1:5000 dilution and a TMB substrate. The platewas read on a Molecular Device plate reader with end point assay at OD450 nm. As shown in FIGS. 4, 5 and 6, in the ELISA binding assay,greater than 50% of the scFvs generated for XB1511 and XB2202 showedspecific binding to PDGFRβ. In contrast, the unpaired VLs alone did notshow binding to PDGFRβ (see FIG. 7).

The affinity of several scFvs was determined by solution basedequilibrium binding assay. Specifically, 120 pmol of scFv RNA wastranslated into free protein with ³⁵S Met incorporated. The translatedreaction mixture was 3-fold diluted in binding buffer containing 1×PBSwith 0.025% triton, 1 mg/mL BSA and 0.1 mg/mL sssDNA. Human PDGFRβ wasdiluted in the same binding buffer to final concentrations from 100 nMto 0 nM. The diluted scFv mixture was incubated with hPDGFRβ in finalvolume of 100 ul on Kingfisher plates (Thermofisher Scientific,97002084). Following incubation, 25 ul of protein A magnetic beads(Invitrogen) were used to capture the PDGFRβ from solution. The capturedPDGFRβ was washed and eluted in kingfisher Reader (ThermofisherScientific). The amount of scFv (labeled with ³⁵S Met) bound to themagnetic bead-immobilzed hPDGFRβ was counted using a scintillationcounter and the Kd was calculated with Graph Pad Prism 5. For theXB1511-derived scFv tested, 2 scFv showed an 8-10 fold higher Kd, 1showed 2.5 fold higher Kd, and 4 showed a similar Kd when compared toXB1511 VH alone (FIG. 8). Only 1 scFv showed a lower K_(D) than XB1511VH alone. As shown in FIG. 9, both of the XB2202-derived scFv testedshowed approximately an 8-10 fold better Kd when compared to XB2202 VHalone.

Example 4. Binding Affinity of Anti-PDGFRβ VH domains to Human and MousePDGFRβ

The R4 framework shuffled human and mouse PDGFRβ enriched VH domain poolselected in Example 2 was cloned into E. coli expression vectors,produced and purified. The binding kinetics of the VH domains to humanand mouse PDFGR was determined using surface plasmon resonance on aBiacore T100. Briefly, human and mouse PDGFR-hIgG1-Fc chimeric fusionprotein were separately immobilized using a Series CM5 sensorchip (CM5)coupled to anti-hIgG1 Fc monoclonal antibody. For each cycle, the PDGFRfusion protein was first captured, followed by the injection of VH for115 seconds at a flow rate of 100 uL/min (association). Immediatelyfollowing the association phase is a dissociation phase of 600 seconds.The surface was regenerated at each cycle with a single injection of 3MMgCl2 (10 uL/min, 60 seconds). Multiple concentrations of VH domain wereinjected (0.55 nM-40 nM) and the resulting sensorgram were analyzed withT100 Evaluation software. The binding kinetics was determined using 1:1binding curve fitting. The binding kinetics of VH domain clones XB2202and XB2708 to human and mouse PDGFRβ are shown in FIGS. 10, 11A and 11B,and 12A and 12B, respectively. These results show that XB2202 and XB2708have a 50-150 fold affinity improvement compared to parental XB1511.Specifically, XB2202 and XB2708 have Kds of 249 pM and 93 pM,respectively and off rates (Koff) of 1.86×10⁻³ and 9.267×10⁻⁴,respectively. Both XB2202 and XB2708 bound to human and mouse PDGFRβ. Itis of particular note that, although they shared the same HCDR3, XB2202was derived from a VH1 family germline sequence and XB2708 was derivedfrom VH3 family germline sequence.

Example 5. Inhibition of PDGFBB Binding to PDGFRβ

The ability of the XB2202 VH domain, disclosed herein, to antagonize thebinding of PDGFBB ligand to the human PDFGRb was assessed using surfaceplasmon resonance on a Biacore T100. Briefly, human PDGFR-hIgG1-Fcchimeric fusion protein was immobilized using a Series CM5 sensorchipcoupled with anti-hIgG1 Fc monoclonal antibody. 10 nM of human PDGFBBwas injected to pre-captured human PDGFRβ obtain the 100% bindingresponse unit to PDGFRβ in the absence VH. For each successive cycle,the PDGFR fusion protein was first captured then VH domain was theninjected for 120 seconds. After washing away unbound VH domain, 10 nM ofPDGFBB was then injected for 120 seconds. The surface was regenerated ateach cycle with a single injection of 3M MgCl2 (10 uL/min, 60 seconds).Multiple concentrations of VH were injected (0.46 nM-60 nM), theresulting sensorgram were analyzed with T100 Evaluation software, andthe PDGFBB binding inhibition was calculated. As shown in FIG. 13,XB2202 inhibits PDGFBB binding to human PDFGRb with an IC50 of less than5 nM.

Example 6. Inhibition of Pericyte Cell Migration

The ability of the XB2708 VH domain, disclosed herein, to antagonizePDGF-BB induced pericyte migration in vitro was determined. Primaryhuman retinal pericytes were obtained from Cell Systems Corporation(Kirkland, Wash.) and cultured according to the manufacturer'ssuggestions using CSC full growth medium. Approximately 125 cells (2-5passages) were seeded in each well of 384-well BIND© biosensor platescoated with human plasma fibronectin (5 μg/ml in PBS) and blocked withBSA (1% in PBS) in serum-free medium containing 0.1% BSA. Cells wereallowed to adhere and then serum-starve overnight. Following serumstarvation, cells were incubated for 1 hour with various concentrationsof VHs against human PDGFRβ receptor in a tissue culture incubator.Migration was stimulated at the end of antibody pre-incubation byPDGF-BB addition to a final concentration of 5 ng/ml in serum-freemedium. Well images were acquired using a BIND© Scanner every 18 minutesfor 20 hours in a tissue culture incubator at 37° C. with 5% CO₂and >75% humidity. Collected data were analyzed using a Matlab-basedcentroid identification and tracking algorithm to calculate the speed ofcells between the hours 10 and 16. The results set forth in FIG. 14 showthat XB2708 can antagonize PDGF-BB-induced pericyte migration with anIC50 of 0.54 nM.

Example 7. Conversion of VH-VL Pairs to Heterotetrameric IgG andDemonstration of Biological Activity

XB1511 VH and D8 VL were expressed together in a heterotetrameric IgG in293T cells. Cell culture supernatant was collected after 48 hours and 96hours and the expressed IgG was purified with protein A agarose beads.The IgG was produced at 8 mg/L without any optimization. To evaluate thebiological activity of the XB1511/D8 IgG, HFF-1 human foreskinfibroblasts were seeded in 384-well BIND biosensors and allowed toattach overnight in serum-free media. The fibroblast cells were thenstimulated with 5 ng/mL or 10 ng/mL of PDGFBB ligand and allowed tomigrate for 18 hours in the presence or absence of 100 nM XB1511/D8 IgG.BIND Scanner images were captured every 15 minutes and software analysistools used to measure the track lengths of individual cell migrationresponses. Track length is represented by a “heat map” from blue (nomigration) to red (maximal migration). As shown in FIG. 15, theXB1511/D8 IgG was able to completely block the PDGFBB-induced migrationof human fibroblasts.

Example 8. scFv Thermostability

The thermostability of the XB2202 VH and XB2202/A4 scFv were determined.Specifically, 1 mg/mL of XB2202 and XB2202-A4 were incubated at 4° C.,37° C., 60° C. and 70° C. for 12 hours and a PDGFRβ binding ELISA wasperformed to test the binding activity of the protein after incubation.As shown in FIG. 16, the XB2202 VH domain lost significant PDGFRβbinding activity after incubation at 60° C. and completely lost bindingactivity after incubation at 70° C. The Tm of XB2202 was measured to beapproximately 62° C. In contrast, the XB2202/A4 scFv was completelyactive after 12 hour incubation at 70° C., indicating that the Tm of theXB2202 scFv was greater than 70° C.

Example 9. Expression, Purification and Concentration of IgG1 Antibodies

XB1511/D8 and XB2202/A4 VH/VL pairs were separately expressed asfull-length heterotetrameric IgG1 antibodies in 293T cells and purified.The amino acid sequences of the heavy and light chains of XB1511/D8 andXB2202/A4 IgG1 antibodies are set forth in Table 7, herein.

Cell culture supernatants were obtained by filtration and expressedantibodies purified using a two-step purification scheme. Specifically,Protein A affinity purification was performed, with antibody boundelution at pH3.5. The pH of the Protein A eluate was adjusted to pH 7using 1M Tris, and purified further by ion exchange chromatography usinga HiTrap Q XL column (GE Healthcare). The purified antibody was storedin PBS at pH7.

TABLE 7Amino acid sequences of XB1511/D8 and XB2202/A4 VH/VL pairs formattedas full-length heterotetrameric IgG1 antibodies. AntibodyAmino Acid Sequence SEQ ID chain (Signal sequences underlined) NO.XB1511 GWSLILLFLVAVATRVLSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIS 420 IgG1WVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAIHGGDRSYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK D8DFQVQIISFLLISASVIMSRGEIVMTQSPGTLTLSPGEGATLSCRASQSVTSN 421 CkappaYLAWYQQRPGQAPRLLIYDASNRATGIPDRFSGSGFGTDFTLTISRLEPEDFAVYYCQQYVNSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC XB2202GWSLILLFLVAVATRVLSQVQLVQSGAEVKKPGSSVRVSCKASGGTFSRHAIS 422 IgG1WVRQAPGQGLEWIGGILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK A4DFQVQIISFLLISASVIMSRGDVVMTQSPSSLSASVGDRVTITCQASQDISNW 423 CkappaLNWYQQKPGKAPKLLIYEASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYNNVLRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVOLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The antibody expression level and the antibody concentration after eachpurification step was determined by measuring the A₂₈₀ of the antibodysolution. The purity and quality of the purified antibodies wasdetermined by size-exclusion high-performance liquid chromatography(SEC-HPLC). The results of these experiments are set forth in Table 8,herein. These data show that when XB1511/D8 and XB2202/A4 VH/VL pairsare formatted as full-length heterotetrameric IgG1 antibodies, theresultant antibodies are highly manufacturable in that they areexpressed at high levels, are easily purified to a high purity, andexhibit little aggregation.

TABLE 8 Analysis of expression and purification of XB1511/D8 andXB2202/A4 IgG1 antibodies. Amount of Amount of antibody Antibody %Antibody antibody after ion- recovery Culture Expression after proteinexchange after 2 step Antibody % antibody Antibody volume level Apurification purification purification % purity aggregates XB1511/D8IgG1 2.0 L 24 mg/L 46 mg 44 mg 97.8% 95.8% 2.4% XB2202/A4 IgG1 1.4 L 47mg/L 62 mg 54 mg  87% 96.9% 2.5%

The purified XB1511/D8 and XB2202/A4 IgG1 antibodies were furtheranalysed for their ability to be concentrated. Specifically, solutionsof each antibody were concentrated to 50 mg/ml using centricon ultrafiltration spin columns with 10 kDa and 30 kDa cut-off limits. Theintegrity of the concentrated solution was analyzed by SEC-HPLC. Fromthis analysis it was determined that the 50 mg/ml solution of XB1511/D8IgG1 had a purity of about 96% and contained about 2.4% of antibodyaggregates, whilst the 50 mg/ml solution of XB2202/A4 IgG1 had a purityof about 97.8% and contained about 2.2% of antibody aggregates. Thesedata demonstrate that XB1511/D8 and XB2202/A4 IgG1 antibodies are highlystable in a concentrated solution.

Example 10. Thermostability of XB1511/D8 and XB2202/A4 IgG1 Antibodies

The thermostability of XB1511/D8 and XB2202/A4 IgG1 antibodies weredetermined using a fluorescence based assay. Specifically, 5 mg/ml ofpurified XB1511/D8 IgG1, XB2202/A4 IgG1, or human IgG1 control weremixed with Sypro orange dye (Sigma) and the temperature of the mixtureincreased in 1 degree increments from 25° C. to 95° C. The Sypro orangedye incorporates into the IgG when the temperature increases and the IgGunfolds. The fluorescent signal produced by the association of Syproorange dye with the IgGs was monitored using a BioRad CFX96 instrument.In this assay, the negative regression of the Sypro orange signal wasused to identify the peak melting (i.e. T_(m)) point for each protein.

From this analysis it was determined that XB1511/D8 and XB2202/A4 havemelting temperatures (T_(m)) of 67° C. to 70° C., respectively. Thiscompared well to the human IgG1 control antibody which exhibited a Tm of72° C. This data demonstrate that the VH and VH/VL pairs of theinvention are capable of being formatted into highly thermostablefull-length IgG molecules.

Example 11. Binding Affinities of XB2202 VH, scFv and IgG1 Antibodies toHuman

The binding kinetics of XB2202 VH domain, XB2202/A4 scFv and XB2202/A4IgG1 to human PDFGR were determined using surface plasmon resonance on aBiacore T100. Briefly, recombinant human PDGFR-hIgG1-Fc chimeric fusionprotein (R&D, #385-PR-100/CF) was immobilized on a Series CM5 sensorchipcoupled with anti-hIgG1 Fc monoclonal antibody (for the VH and ScFvassays) or anti-6His antibody (for the IgG assay). XB2202 VH domain,XB2202/A4 scFv and XB2202/A4 IgG1 were flown over the surface at 50 or100 ul/min for 3 min at different concentrations (75, 50, 25, 10, 5, and1 nM) and allowed to dissociate for 10 min. The data was analyzed usingthe Biacore T100 analysis software using a 1:1 model. Mass transport waschecked and avoided to allow accurate measurements. All data was doublereferenced according to Biacore standard protocol.

The binding kinetics of XB2202 VH domain, XB2202/A4 scFv and XB2202/A4IgG1 antibodies to human PDGFRβ are shown in Table 9, herein. These datashow that XB2202 VH domain, XB2202/A4 scFv and XB2202/A4 IgG1 each havea high binding affinity for PDGFRβ. It is of particular note thatXB2202/A4 scFv and XB2202/A4 IgG1 exhibit an improved off-rate(1.54×10⁻¹ s⁻¹ and 1.56×10⁻¹ s⁻¹, respectively) compared to that of theunpaired XB2202 VH domain alone (2.95×10⁻³ s⁻¹).

TABLE 9 Binding Kinetics of XB2202 VH domain, XB2202/A4 scFv andXB2202/A4 IgG1 to human PDGFRβ Antibody On-rate (M⁻¹s⁻¹) Off-rate (s⁻¹)Kd (M) XB2202 VH 1.30 × 10⁷ 2.95 × 10⁻³  2.27 × 10⁻¹⁰ XB2202/A4 ScFv7.06 × 10⁵ 1.54 × 10⁻³ 2.18 × 10⁻⁹ XB2202/A4 IgG1 9.80 × 10⁵ 1.56 × 10⁻³1.59 × 10⁻⁹

Example 12. Functional Analysis of Anti-PDGFRβ Antibodies Using In VivoMouse Models

The ability of the anti-PDGFRβ antibodies disclosed herein to inhibitPDGF-induced vascularization in vivo is evaluated using the developingretina vasculature model, the corneal neovascularization model, and/orthe choroidal neovascularization model described in Nobuo et al. Am. J.Path, (2006) 168(6), 2036-2052 (which is incorporated by referenceherein in its entirety). In these assays antibodies are administered tomice as VH domains, scFv, and/or full length IgG.

1.-39. (canceled)
 40. A diverse library of unpaired VH domains, whereineach member of the library binds to human PDGFRβ, and wherein thelibrary is a nucleic acid display library.
 41. The library of claim 40,wherein the nucleic acid display library is a DNA display library. 42.The library of claim 40, wherein diversity lies in the FR1-FR3 regions,and wherein each member of the library comprises the CDR3 amino acidsequence set forth in SEQ ID NO:
 1. 43. A diverse library of stableVH/VL pairs, wherein each member of the library binds to human PDGFRβ,and wherein the library is a nucleic acid display library.
 44. Thelibrary of claim 43, wherein the nucleic acid display library is a DNAdisplay library.
 45. The library of claim 43, wherein each member of thelibrary comprises a VH domain comprising the CDR3 amino acid sequenceset forth in SEQ ID NO:
 1. 46. The library of claim 45, wherein the VLdomains are human VL domains.